CN109536527A - A kind of new method of point mutation reparation - Google Patents
A kind of new method of point mutation reparation Download PDFInfo
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- CN109536527A CN109536527A CN201811428895.3A CN201811428895A CN109536527A CN 109536527 A CN109536527 A CN 109536527A CN 201811428895 A CN201811428895 A CN 201811428895A CN 109536527 A CN109536527 A CN 109536527A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/102—Mutagenizing nucleic acids
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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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- C12N2800/00—Nucleic acids vectors
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- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
Abstract
The present invention provides a kind of new method of point mutation reparation, key step is as follows: in target gene group sequence, selected cytogene mutational site is the gene loci repaired；According to selected gene repair site, the sgRNA of CRISPR/Cas system is designed；SgRNA is building up on the expression vector for carrying CRISPR/Cas9 albumen；One section of correct sequence is chosen near the upstream or downstream in mutational site, and thus designs Donor DNA, the template as gene mutation reparation；The cell of in vitro culture carrying mutational site gene；Using the method for electrotransfection, the expression vector of sgRNA and CRISPR/Cas9, Donor DNA are transfected into cell.The present processes are by Cas9 expression vector, sgRNA, and Donor DNA and the Apoptosis for the carrying Msh5 point mutation established in vitro are sufficiently mixed, then electrotransfection processing is carried out to cell line, increase the transfection efficiency of cell, and this mutational site is repaired using CRISPR/Cas9 system, to provide scientific basis for treatment female primary amenorrhoea disease.
Description
Technical field
The invention belongs to gene repair technical fields, more particularly, to a kind of new method of point mutation reparation.
Background technique
CRISPR/CAS9 system is the new high efficiency gene editing technique of one kind developed in recent years, it can be thin
Born of the same parents' level is deleted genome, is modified.After CRISPR/CAS system is announced at the beginning of 2013, quickly just in whole world model
It is had been widely used in enclosing, and one of ten big sciences breakthrough in 2015 is chosen as by " SCIENCE " magazine.CRISPR/
CAS9 is a kind of complex of RNA- protein, and the most commonly used is CAS9 nucleases, are made of 1409 amino acid, have 2 it is important
Nuclease domain, be RUVC and HNH structural domain respectively.It is single-stranded by the complementary DNA of targeting that HNH structural domain is responsible for fracture,
Cleavage site is located at PAM (PROTOSPACER-ADJACENT MOTIFS) Sequences upstream 3NT.RUVC structural domain is responsible for fracture
Another DNA chain, cleavage site are located at 3~8NT of PAM Sequences upstream.RNA-DNA double-strand initially forms in the site PAM.CR-
RNA/TRANCR-RNA is incorporated near PAM sequence, and the activity of CAS9 nuclease is induced by RUVC and HNH structural domain.Mesh
The preceding method by genetic engineering by both RNA of CR-RNA and TRACR-RNA, is simplified to the same function
sgRNA。
Using CRISPR/CAS9 system to mutated gene edited in field of medical research application it is rather extensive,
The treatment of HIV, phene therapy, such as the choreic treatment of Huntington, treatment of cancer, hematologic disease aspect and eye disease
Etc. treatment all achieve significant progress, open new gate for the research of disease.
Our early-stage study has found a kind of genetic mutation (TING GUO ET that can lead to female primary amenorrhoea
AL, 2017, HUMAN MOLEC Μ LAR GENETICS), which is located at the mutation of G to the C in 1459 site of Msh5 gene, should
Mutation causes Msh5 to be unable to normally travel function, so that reproduction cell meiosis process cannot be completed, finally causes to give birth to
Cell colonization is dead.
Summary of the invention
In view of this, the present invention is directed to propose a kind of new method of point mutation reparation, utilizes electrotransfection technology and height
The treatment method that the gene editing technology CRISPR/Cas9 system of effect combines repairs reproduction cell in vitro.
In order to achieve the above objectives, the technical scheme of the present invention is realized as follows:
A kind of new method of point mutation reparation, key step are as follows:
(1) in target gene group sequence, selected cytogene mutational site is the gene loci repaired；
(2) according to selected gene repair site, the sgRNA of CRISPR/Cas system is designed；
(3) sgRNA is building up on the expression vector for carrying CRISPR/Cas9 albumen；
(4) one section of correct sequence is chosen near the upstream or downstream in mutational site, and thus designs Donor DNA,
Template as gene mutation reparation；
(5) in vitro culture carries the cell of mutational site gene；
(6) method for utilizing electrotransfection, the expression vector of sgRNA and CRISPR/Cas9, Donor DNA are transfected into carefully
In born of the same parents.
Further, the mutational site of selected genes is Msh5 point.
Further, which is present in female reproductive cell.
Further, sgRNA sequence corresponding to the mutational site Msh5 are as follows:
Guide#1:CTATCGTAGCGCCCGGACCAAGG
Guide#8:CAAGGAGCTGTACACGCTGCTGG.
Further, the upstream primer and downstream primer sequence of Guide#1 is respectively as follows:
F-CACCGCTATCGTAGCGCCCGGACCA
R-AAACTGGTCCGGGCGCTACGATAGC；
The upstream primer and downstream primer sequence of Guide#8 is respectively as follows:
F-CACCGCAAGGAGCTGTACACGCTGC
R-AAACGCAGCGTGTACAGCTCCTTGC。
Further, the expression vector of CRISPR/Cas9 albumen is pCS (eGFP) carrier.
Further, the sequence of Donor DNA are as follows:
CAGTTTCTCTCAGAGGACAAGCTGCACTATCGTAGCGCCCGGACCAAGGAGCTGGACACGCTGCTGGG
AGACCTGCACTGCGAGATCCGGGGTGAGGAGCCCGTGGTAGGAGGGGGCAGGCTGCTCTAAC。
Further, the pulse power voltage of electrotransfection be 33V, pulse spacing 50ms~950ms, forward and reverse each 4 times.
It should be noted that being clinically abnormal there are many reproduction cell is all as caused by gene mutation, originally
The method of application is that Cas9 expression vector, sgRNA and Donor DNA are carried the small of Msh5 point mutation with what is established in vitro
Mouse fibroblast cell system is sufficiently mixed, and then carries out electrotransfection processing to cell line, increases the transfection efficiency of cell, and utilize
CRISPR/Cas9 system repairs this mutational site.By it is this processing so that Cas9 expression vector, sgRNA and
Donor DNA enters in reproduction cell, so that the gene mutation site in reproduction cell be repaired, and utilizes the side of sequencing
Method detects the efficiency that mutational site is repaired, to provide scientific basis for treatment female primary amenorrhoea disease.
Detailed description of the invention
Fig. 1 is that forward primer sequencing result is sequenced in Extag PCR product；
Fig. 2 is that reverse primer sequencing result is sequenced in Extag PCR product；
Fig. 3 is T7EI endonuclease reaction system electrophoresis detection result.
Specific embodiment
In addition to being defined, technical term used in following embodiment has universal with those skilled in the art of the invention
The identical meanings of understanding.Test reagent used in following embodiment is unless otherwise specified conventional biochemical reagent；It is described
Experimental method is unless otherwise specified conventional method.
Below with reference to embodiment, the present invention will be described in detail.
It is as follows to the specific research approach of Msh5 point mutation reparation for carrying the mouse of Msh5 point mutation:
The building of one carrier of embodiment
1, the design of sgRNA: using sgRNA Photographing On-line tool, chooses suitable sgRNA in the location proximate of mutation.
SgRNA selects Guide#1 and Guide#8, sequence information are as follows:
Guide#1 CTATCGTAGCGCCCGGACCAAGG
Guide#8 CAAGGAGCTGTACACGCTGCTGG。
2, sgRNA is building up on pCS (eGFP) carrier, which carries Cas9 albumen.
(1) restriction enzyme site BbsI, primer sequence design primer: are introduced in the primer of upstream and downstream are as follows:
Guide#1:F-CACCGCTATCGTAGCGCCCGGACCA
R-AAACTGGTCCGGGCGCTACGATAGC
Guide#8:F-CACCGCAAGGAGCTGTACACGCTGC
R-AAACGCAGCGTGTACAGCTCCTTGC
(2) primer annealing is at double-strand: primer is annealed after being dissolved as 200 μM with TE
Annealing system totally 20 μ L
It after mixing, is directly placed into boiling water and anneals, until being cooled to room temperature, be put in 4 DEG C.
(3) T4 ligase connects
37 DEG C of the BbsI restriction enzyme overnight digestions of pCS (eGFP) carrier, and gel extraction obtains the load of linearisation
Body, this carrier is for connecting；Product after taking 1 μ L to anneal adds 99 μ L ddH2O to be diluted to 100 μ L, and dilution is for connecting.
Linked system totally 15 μ L
After mixing, 16 DEG C of connections are overnight.
(4) it converts
Take 5 μ L connection products in 50 μ L competence, on ice stand 25 minutes, 42 DEG C heat shock 30 seconds, on ice stand 2 points
Clock.The LB of 450 μ L nonreactives is added, 37 DEG C, 200rpm, recovers 1 hour.
(5) coated plate
Bacterium solution after taking 100 μ L to recover is applied to Amp+Plate in, 37 DEG C of overnight incubations.
(6) picking monoclonal extracts plasmid with the small extraction reagent kit of plasmid, and whether sequence verification carrier correctly connects.
The cutting efficiency of two carrier of embodiment detects
One, Guide#1 cutting efficiency detects
The carrier of building endotoxin-free plasmid extracts kit is extracted into plasmid.
1, fertilized eggs procaryotic injection carries out blastaea identification, verifies cleavage activity
Vector injection is entered into protokaryon phase fertilized eggs, to development of fertilized ova to blastula stage (4 days or so), carries out blastaea identification,
It detects whether to have carried out correct cutting.99 μ L lysates, 1 μ L Proteinase K are added in blastaea, and 56 DEG C of water-baths crack overnight.Cracking
Product afterwards is handled 5 minutes in 95 DEG C, is denaturalized Proteinase K, product can be used for PCR.Blastaea identification carries out PCR using Extag
Amplification, send the PCR product for having purpose band to sequencing.
50 μ L of Extag PCR system
Extag PCR response procedures
PCR product sequencing, sequencing result is specifically shown in Fig. 1 and Fig. 2, wherein showing, starts to occur in theoretical cleavage site
Peak is covered, illustrates there is cleavage activity.
2, F9 cell is transfected, cutting efficiency is detected
The F9 cell of 24 orifice plates is transfected with Lipo3000.The amount of transfected plasmids is 500ng, and untransfected group compares.Transfection
After 48 hours, cell is collected, lysate is added, extracts DNA.After extracting DNA, T7EI digestion and TA clone are carried out, detection is cut
Cut efficiency.
(1) T7EI digestion
1. PCR amplification
Amplimer F:CCCAAGGGATGAAAAGCCAC
R:GGGAGAGTAATGCGGTCTCG
2. PCR product is after purification, it is mixed in EP pipe according to following system:
3. heat denatured, renaturation process of annealing
Boiling water is added in 1 liter of beaker, EP pipe is put into boiling water, and beaker is cooling in room temperature.
4. T7EI endonuclease reaction
0.5 μ L T7EI enzyme is added in above-mentioned reaction system, 37 DEG C, reacts 30 minutes.2 μ L DNA are added immediately
loading buffer(6X).After mixing, 65 DEG C are boiled 10 minutes.The detection of 2% agarose electrophoresis, electrophoresis result are as shown in Figure 3.
PCS (eGFP) represents transfection zero load in figure, and 1,2,3 respectively represent three of transfection pCS (eGFP)+gRNA1# carrier
Parallel hole.By gray analysis, mutation band gray scale/wild type band gray scale is calculated, obtaining final mutation rate is about 12%.
(2) TA is cloned: using pEASY-T1simple cloning kit
1. PCR amplification
Amplimer F:CCCAAGGGATGAAAAGCCAC
R:GGGAGAGTAATGCGGTCTCG
2. PCR product is connect with pEASY-T1simple cloning vector
Linked system
Reaction condition: system is gently mixed, and is reacted 10 minutes for 25 DEG C in PCR
3. converting
Connection product in 50 μ L competence, on ice stand 25 minutes, 42 DEG C heat shock 30 seconds, on ice stand 2 minutes.It is added
The LB of 450 μ L nonreactives, recovers 1 hour by 37 DEG C, 200rpm.
4. coated plate
5. positive colony detects
Bacterium solution PCR identification: PCR identification is carried out with carrier universal primer M13F and M13R
Positive band send sequencing: 60 single colonies of picking, positive band is 20, and sequencing result is shown, only 3
It is cut in theoretical site, efficiency is about 15%.
Embodiment three designs Doner DNA
One section of correct sequence is chosen near the upstream and downstream in mutational site as Donor, and raw work is sent to synthesize.Sequence
Are as follows:
CAGTTTCTCTCAGAGGACAAGCTGCACTATCGTAGCGCCCGGACCAAGGAGCTGGACACGCTGCTGGG
AGACCTGCACTGCGAGATCCGGGGTGAGGAGCCCGTGGTAGGAGGGGGCAGGCTGCTCTAAC。
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of new method of point mutation reparation, which is characterized in that key step is as follows:
(1) in target gene group sequence, selected cytogene mutational site is the gene loci repaired；
(2) according to selected gene repair site, the sgRNA of CRISPR/Cas system is designed；
(3) sgRNA is building up on the expression vector for carrying CRISPR/Cas9 albumen；
(4) one section of correct sequence is chosen near the upstream or downstream in mutational site, and thus designs Donor DNA, as
The template of gene mutation reparation；
(5) in vitro culture carries the cell of mutational site gene；
(6) method for utilizing electrotransfection, the expression vector of sgRNA and CRISPR/Cas9, Donor DNA are transfected into cell.
2. a kind of new method of point mutation reparation according to claim 1, which is characterized in that the mutation of selected genes
Site is Msh5 point.
3. a kind of new method of point mutation reparation according to claim 2, which is characterized in that the Msh5 point is present in
In female reproductive cell.
4. a kind of new method of point mutation reparation according to claim 1, which is characterized in that the mutational site Msh5 institute
Corresponding sgRNA sequence are as follows:
Guide#1:CTATCGTAGCGCCCGGACCAAGG
Guide#8:CAAGGAGCTGTACACGCTGCTGG.
5. a kind of new method of point mutation reparation according to claim 4, which is characterized in that
The upstream primer and downstream primer sequence of Guide#1 is respectively as follows:
F-CACCGCTATCGTAGCGCCCGGACCA
R-AAACTGGTCCGGGCGCTACGATAGC；
The upstream primer and downstream primer sequence of Guide#8 is respectively as follows:
F-CACCGCAAGGAGCTGTACACGCTGC
R-AAACGCAGCGTGTACAGCTCCTTGC。
6. a kind of new method of point mutation reparation according to claim 1, which is characterized in that CRISPR/Cas9 egg
White expression vector is pCS (eGFP) carrier.
7. a kind of new method of point mutation reparation according to claim 1, which is characterized in that
The sequence of Donor DNA are as follows:
CAGTTTCTCTCAGAGGACAAGCTGCACTATCGTAGCGCCCGGACCAAGGAGCTGGACACGCTGCTGGGAGAC
CTGCACTGCGAGATCCGGGGTGAGGAGCCCGTGGTAGGAGGGGGCAGGCTGCTCTAAC。
8. a kind of new method of point mutation reparation according to claim 1, which is characterized in that the pulse electricity of electrotransfection
Source voltage be 33V, pulse spacing 50ms~950ms, forward and reverse each 4 times.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251419A (en) * | 2019-11-07 | 2021-01-22 | 青岛清原化合物有限公司 | Method for generating new mutation in organism and application |
| CN112980880A (en) * | 2021-03-08 | 2021-06-18 | 浙江大学 | Method for constructing Fzd6-Q152E site-directed mutagenesis mouse model based on CRISPR/Cas9 and application |
| WO2021208937A1 (en) * | 2020-04-14 | 2021-10-21 | Tsinghua University | Template-independent genome editing and repairing correct frame-shifting disease in vivo |
Citations (1)
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| WO2016176191A1 (en) * | 2015-04-27 | 2016-11-03 | The Trustees Of The University Of Pennsylvania | Dual aav vector system for crispr/cas9 mediated correction of human disease |
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2018
- 2018-11-27 CN CN201811428895.3A patent/CN109536527A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016176191A1 (en) * | 2015-04-27 | 2016-11-03 | The Trustees Of The University Of Pennsylvania | Dual aav vector system for crispr/cas9 mediated correction of human disease |
Non-Patent Citations (2)
| Title |
|---|
| HAO YIN等: "Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype", 《NATURE BIOTECHNOLOGY》 * |
| 郭婷: "DNA损伤修复相关基因CSB-PGBD3、MSH5和FMHR1在卵巢早衰发病中的作用机制研究", 《中国博士学位论文全文数据库》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251419A (en) * | 2019-11-07 | 2021-01-22 | 青岛清原化合物有限公司 | Method for generating new mutation in organism and application |
| CN112251419B (en) * | 2019-11-07 | 2023-11-10 | 青岛清原化合物有限公司 | Method for generating new mutation in organism and application thereof |
| WO2021208937A1 (en) * | 2020-04-14 | 2021-10-21 | Tsinghua University | Template-independent genome editing and repairing correct frame-shifting disease in vivo |
| CN112980880A (en) * | 2021-03-08 | 2021-06-18 | 浙江大学 | Method for constructing Fzd6-Q152E site-directed mutagenesis mouse model based on CRISPR/Cas9 and application |
| CN112980880B (en) * | 2021-03-08 | 2023-05-02 | 浙江大学 | Method for constructing Fzd6-Q152E fixed-point mutation mouse model based on CRISPR/Cas9 and application |
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