CN110272921A - A kind of safe and efficient CRISPR/Cas9 gene editing method - Google Patents
A kind of safe and efficient CRISPR/Cas9 gene editing method Download PDFInfo
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- CN110272921A CN110272921A CN201910536641.1A CN201910536641A CN110272921A CN 110272921 A CN110272921 A CN 110272921A CN 201910536641 A CN201910536641 A CN 201910536641A CN 110272921 A CN110272921 A CN 110272921A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/22—Ribonucleases RNAses, DNAses
Abstract
The present invention relates to gene engineering technology fields, especially a kind of safe and efficient CRISPR/Cas9 gene editing method, the following steps are included: S1, being determined to the target-gene sequence on target gene, and open the DNA double spiral of target gene, using Cas9 vitro enzyme to it into shearing, the DNA sequence dna of target gene causes to be broken, and knockout goes genetic fragment in spite of illness；S2, sgRNA sequence is designed, and carried out artificial synthesized；S3, the bacterium for choosing competence, thaw to it；S4, strain is inoculated into BL solution, keeping temperature is 37 DEG C, is cultivated in the environment that revolving speed is 250r/min, and stayed overnight；S5, picking monoclonal, using alkaline lysis method of extracting plasmid；The DMEM culture medium of S6, absorption serum-free and antibiotic are added 6 μ l transfection reagent PEI and piping and druming are uniformly mixed into sterile centrifuge tube, and 2 μ g Plasmid DNA of addition are uniformly mixed, and temperature control is 37 DEG C.The present invention has the characteristics that at low cost, simple for production, quickly and efficiently.
Description
Technical field
The present invention relates to gene engineering technology field more particularly to a kind of safe and efficient CRISPR/Cas9 gene editings
Method.
Background technique
CRISPR-Cas9 is a kind of adaptive immunity defence that bacterium and archeobacteria are formed during long-term evolution, can
For fighting the virus and exogenous DNA of invasion.And CRISPR-Cas9 gene editing technology, then it is specific to target gene progress
The technology of DNA modification, this technology are also the method currently used for forward position in gene editing.With the base on the basis CRISPR-Cas9
Because editing technique all shows great application prospect in the application field that series of genes is treated, for example, blood disease, tumour and
Other genetic diseases, so a kind of it is proposed that safe and efficient CRISPR/Cas9 gene editing method.
Summary of the invention
The purpose of the present invention is to solve disadvantages existing in the prior art, and the one kind proposed is safe and efficient
CRISPR/Cas9 gene editing method.
To achieve the goals above, present invention employs following technical solutions:
Design a kind of safe and efficient CRISPR/Cas9 gene editing method, comprising the following steps:
S1, the target-gene sequence on target gene is determined, and opens the DNA double spiral of target gene, used
To it into shearing, the DNA sequence dna of target gene causes to be broken Cas9 vitro enzyme, and knockout goes genetic fragment in spite of illness；
S2, design sgRNA sequence, and carry out artificial synthesized, be then combined sgRNA and Cas9, and to DNA into
Row is repaired, incubation at room temperature 10min；
S3, the bacterium for choosing competence, thaw to it, take the DNA repaired in S2 that 10 μ l are added, are added to 50
In the competent cell of μ l, it is uniformly mixed, is put into and stands 30min on ice；
S4, strain is inoculated into BL solution, keeping temperature is 37 DEG C, it is cultivated in the environment that revolving speed is 250r/min, and
It is stayed overnight；
S5, picking monoclonal, using alkaline lysis method of extracting plasmid, and are detected；
6 μ l transfection reagent PEI are added into sterile centrifuge tube in the DMEM culture medium of S6, absorption serum-free and antibiotic
And blow and beat and be uniformly mixed, room temperature 10min, and 2 μ g Plasmid DNA are added and are uniformly mixed, it is placed at room temperature for 15min, then by mixed liquor
Cell culture is added to be uniformly mixed, temperature control is 37 DEG C, and culture solution is added and is mixed.
Preferably, the culture solution 4-6h in cell culture is replaced, and culture medium is given birth to by balance salt water, amino acid, dimension
Element, carbohydrate, inorganic ions, calf serum composition.
Preferably, when extracting plasmid to alkaline lysis, comprising the following steps:
A, choose the bacterium solution in S4, be placed in centrifuge tube, be then added sodium hydroxide solution, and to centrifuge tube it is upper and lower into
Row is reverse；
B, acetic acid first solution is then added, turn upside down shaking 8s, is subsequently placed in 3-5min on ice, is then added
Detergent SDS carries out centrifugal treating to it with centrifuge, and chromosomal DNA has from solution with denatured protein and cell fragment
Precipitate to effect；
C, supernatant is then taken out, is then added isopropanol in supernatant, Plasmid DNA can generate precipitating, then by plasmid
DNA takes out.
Preferably, the bacterium of the competence of selection is Escherichia coli.
Preferably, sgRNA length is less than 80bp.
Preferably, the bacterium of competence produce the following steps are included:
1), by cell be added fluid nutrient medium in, in 37 DEG C of culture 1h, transfer it on 37 DEG C of shaking tables with
200r/min speed culture 2-3h is centrifuged 2min at 4 DEG C with the speed of 8000r/min；
2) it, then prepared by CaCl2 solution, and sterilized to CaCl2 solution at 200-300 DEG C of temperature, then
Natural cooling is carried out to place it in 4-6min on ice after the completion of cooling and be eventually adding CaCl2 solution, and be kept on ice
30min obtains competent cell.
A kind of safe and efficient CRISPR/Cas9 gene editing method proposed by the present invention, beneficial effect are: have at
This is low, simple for production, quickly and efficiently the characteristics of, while sgRNA is designed, increases the stability of sgRNA, reduce simultaneously
The possibility that sgRNA misses the target.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution in the embodiment of the present invention is clearly and completely retouched
It states, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.
Embodiment 1
A kind of safe and efficient CRISPR/Cas9 gene editing method, comprising the following steps:
S1, the target-gene sequence on target gene is determined, and opens the DNA double spiral of target gene, used
To it into shearing, the DNA sequence dna of target gene causes to be broken Cas9 vitro enzyme, and knockout goes genetic fragment in spite of illness；
S2, design sgRNA sequence, and carry out artificial synthesized, be then combined sgRNA and Cas9, and to DNA into
Row is repaired, and incubation at room temperature 10min, sgRNA length is less than 80bp, increases the stability of sgRNA, while reducing sgRNA and missing the target
Possibility；
The bacterium of S3, the bacterium for choosing competence, the competence of selection are Escherichia coli, thaw, are taken in S2 to it
The DNA that repairs 10 μ l are added, be added in the competent cell of 50 μ l, be uniformly mixed, be put into and stand 30min on ice；
S4, strain is inoculated into BL solution, keeping temperature is 37 DEG C, it is cultivated in the environment that revolving speed is 250r/min, and
It is stayed overnight；
S5, picking monoclonal, using alkaline lysis method of extracting plasmid, and are detected；
6 μ l transfection reagent PEI are added into sterile centrifuge tube in the DMEM culture medium of S6, absorption serum-free and antibiotic
And blow and beat and be uniformly mixed, room temperature 10min, and 2 μ g Plasmid DNA are added and are uniformly mixed, it is placed at room temperature for 15min, then by mixed liquor
Cell culture is added to be uniformly mixed, temperature control is 37 DEG C, and culture solution is added and is mixed, the culture in cell culture
Liquid 4h is replaced, and culture medium is made of balance salt water, amino acid, vitamin, carbohydrate, inorganic ions, calf serum,
Germiparity proliferation is promoted, ensure that the living environment of culture cell growth and breeding.
When extracting plasmid to alkaline lysis, comprising the following steps:
A, choose the bacterium solution in S4, be placed in centrifuge tube, be then added sodium hydroxide solution, and to centrifuge tube it is upper and lower into
Row is reverse；
B, acetic acid first solution is then added, turn upside down shaking 8s, is subsequently placed in 3-5min on ice, is then added
Detergent SDS carries out centrifugal treating to it with centrifuge, and chromosomal DNA has from solution with denatured protein and cell fragment
Precipitate to effect；
C, supernatant is then taken out, is then added isopropanol in supernatant, Plasmid DNA can generate precipitating, then by plasmid
DNA takes out.
The bacterium of competence produce the following steps are included:
1), by cell be added fluid nutrient medium in, in 37 DEG C of culture 1h, transfer it on 37 DEG C of shaking tables with
200r/min speed culture 2h is centrifuged 2min at 4 DEG C with the speed of 8000r/min；
2) it, then prepared by CaCl2 solution, and sterilized to CaCl2 solution at 200 DEG C of temperature, then carried out
Natural cooling places it in 4-6min on ice and is eventually adding CaCl2 solution, and be kept on ice 30min, obtain after the completion of cooling
Obtain competent cell.
Embodiment 2
A kind of safe and efficient CRISPR/Cas9 gene editing method, comprising the following steps:
S1, the target-gene sequence on target gene is determined, and opens the DNA double spiral of target gene, used
To it into shearing, the DNA sequence dna of target gene causes to be broken Cas9 vitro enzyme, and knockout goes genetic fragment in spite of illness；
S2, design sgRNA sequence, and carry out artificial synthesized, be then combined sgRNA and Cas9, and to DNA into
Row is repaired, and incubation at room temperature 10min, sgRNA length is less than 80bp, increases the stability of sgRNA, while reducing sgRNA and missing the target
Possibility；
The bacterium of S3, the bacterium for choosing competence, the competence of selection are Escherichia coli, thaw, are taken in S2 to it
The DNA that repairs 10 μ l are added, be added in the competent cell of 50 μ l, be uniformly mixed, be put into and stand 30min on ice；
S4, strain is inoculated into BL solution, keeping temperature is 37 DEG C, it is cultivated in the environment that revolving speed is 250r/min, and
It is stayed overnight；
S5, picking monoclonal, using alkaline lysis method of extracting plasmid, and are detected；
6 μ l transfection reagent PEI are added into sterile centrifuge tube in the DMEM culture medium of S6, absorption serum-free and antibiotic
And blow and beat and be uniformly mixed, room temperature 10min, and 2 μ g Plasmid DNA are added and are uniformly mixed, it is placed at room temperature for 15min, then by mixed liquor
Cell culture is added to be uniformly mixed, temperature control is 37 DEG C, and culture solution is added and is mixed, the culture in cell culture
Liquid 6h is replaced, and culture medium is made of balance salt water, amino acid, vitamin, carbohydrate, inorganic ions, calf serum,
Germiparity proliferation is promoted, ensure that the living environment of culture cell growth and breeding.
When extracting plasmid to alkaline lysis, comprising the following steps:
A, choose the bacterium solution in S4, be placed in centrifuge tube, be then added sodium hydroxide solution, and to centrifuge tube it is upper and lower into
Row is reverse；
B, acetic acid first solution is then added, turn upside down shaking 8s, is subsequently placed in 3-5min on ice, is then added
Detergent SDS carries out centrifugal treating to it with centrifuge, and chromosomal DNA has from solution with denatured protein and cell fragment
Precipitate to effect；
C, supernatant is then taken out, is then added isopropanol in supernatant, Plasmid DNA can generate precipitating, then by plasmid
DNA takes out.
The bacterium of competence produce the following steps are included:
1), by cell be added fluid nutrient medium in, in 37 DEG C of culture 1h, transfer it on 37 DEG C of shaking tables with
200r/min speed culture 3h is centrifuged 2min at 4 DEG C with the speed of 8000r/min；
2) it, then prepared by CaCl2 solution, and sterilized to CaCl2 solution at 300 DEG C of temperature, then carried out
Natural cooling places it in 4-6min on ice and is eventually adding CaCl2 solution, and be kept on ice 30min, obtain after the completion of cooling
Obtain competent cell.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (6)
1. a kind of safe and efficient CRISPR/Cas9 gene editing method, which comprises the following steps:
S1, the target-gene sequence on target gene is determined, and opens the DNA double spiral of target gene, using Cas9 body
To it into shearing, the DNA sequence dna of target gene causes to be broken exoenzyme, and knockout goes genetic fragment in spite of illness；
S2, sgRNA sequence is designed, and carries out artificial synthesized, be then combined sgRNA and Cas9, and DNA is repaired
It is multiple, incubation at room temperature 10min；
S3, the bacterium for choosing competence, thaw to it, take the DNA repaired in S2 that 10 μ l are added, are added to 50 μ l's
In competent cell, it is uniformly mixed, is put into and stands 30min on ice；
S4, strain is inoculated into BL solution, keeping temperature is 37 DEG C, is cultivated in the environment that revolving speed is 250r/min, and carry out
Overnight；
S5, picking monoclonal, using alkaline lysis method of extracting plasmid, and are detected；
The DMEM culture medium of S6, absorption serum-free and antibiotic are added 6 μ l transfection reagent PEI and simultaneously blow into sterile centrifuge tube
It beats and is uniformly mixed, room temperature 10min, and 2 μ g Plasmid DNA are added and are uniformly mixed, it is placed at room temperature for 15min, then mixed liquor is added
Cell culture is uniformly mixed, and temperature control is 37 DEG C, and culture solution is added and is mixed.
2. a kind of safe and efficient CRISPR/Cas9 gene editing method according to claim 1, which is characterized in that thin
Culture solution 4-6h in born of the same parents' culture plate is replaced, and culture medium is by balance salt water, amino acid, vitamin, carbohydrate, inorganic
Ion, calf serum composition.
3. a kind of safe and efficient CRISPR/Cas9 gene editing method according to claim 1, which is characterized in that
When extracting plasmid to alkaline lysis, comprising the following steps:
A, the bacterium solution in S4 is chosen, is placed in centrifuge tube, sodium hydroxide solution, and running up and down to centrifuge tube is then added
?；
B, acetic acid first solution is then added, turn upside down shaking 8s, is subsequently placed in 3-5min on ice, be then added in de-sludging
Agent SDS carries out centrifugal treating to it with centrifuge, chromosomal DNA and denatured protein and cell fragment from solution effectively
It precipitates；
C, supernatant is then taken out, isopropanol is then added in supernatant, Plasmid DNA can generate precipitating, then Plasmid DNA is taken
Out.
4. a kind of safe and efficient CRISPR/Cas9 gene editing method according to claim 1, which is characterized in that choosing
The bacterium of the competence taken is Escherichia coli.
5. a kind of safe and efficient CRISPR/Cas9 gene editing method according to claim 1, which is characterized in that
SgRNA length is less than 80bp.
6. a kind of safe and efficient CRISPR/Cas9 gene editing method according to claim 1, which is characterized in that sense
Produced by the bacterium of state the following steps are included:
1), cell is added in fluid nutrient medium, in 37 DEG C of culture 1h, is transferred it on 37 DEG C of shaking tables with 200r/min
Speed culture 2-3h is centrifuged 2min at 4 DEG C with the speed of 8000r/min；
2) it, then prepared by CaCl2 solution, and sterilized to CaCl2 solution at 200-300 DEG C of temperature, then carried out
Natural cooling places it in 4-6min on ice and is eventually adding CaCl2 solution, and be kept on ice 30min, obtain after the completion of cooling
Obtain competent cell.
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Citations (4)
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|---|---|---|---|---|
| CN106566838A (en) * | 2016-11-14 | 2017-04-19 | 上海伯豪生物技术有限公司 | MiR-126 full-length gene knockout kit based on CRISPR-Cas9 technology and application thereof |
| CN107406875A (en) * | 2014-12-20 | 2017-11-28 | 阿克生物公司 | Use the composition and method of CRISPR/Cas systematic proteins targeting abatement, enrichment and segmentation nucleic acid |
| CN108486111A (en) * | 2018-04-04 | 2018-09-04 | 山西医科大学 | The method and its specificity sgRNA of CRISPR-Cas9 targeting knock out people's SMYD3 genes |
| WO2019005851A1 (en) * | 2017-06-26 | 2019-01-03 | Arizona Board Of Regents On Behalf Of Arizona State University | A universal platform to enhance crispr-based gene editing for in vivo therapies |
-
2019
- 2019-06-20 CN CN201910536641.1A patent/CN110272921A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107406875A (en) * | 2014-12-20 | 2017-11-28 | 阿克生物公司 | Use the composition and method of CRISPR/Cas systematic proteins targeting abatement, enrichment and segmentation nucleic acid |
| CN106566838A (en) * | 2016-11-14 | 2017-04-19 | 上海伯豪生物技术有限公司 | MiR-126 full-length gene knockout kit based on CRISPR-Cas9 technology and application thereof |
| WO2019005851A1 (en) * | 2017-06-26 | 2019-01-03 | Arizona Board Of Regents On Behalf Of Arizona State University | A universal platform to enhance crispr-based gene editing for in vivo therapies |
| CN108486111A (en) * | 2018-04-04 | 2018-09-04 | 山西医科大学 | The method and its specificity sgRNA of CRISPR-Cas9 targeting knock out people's SMYD3 genes |
Non-Patent Citations (3)
| Title |
|---|
| PHILIP J.R. ROCHE等: "Double-Stranded Biotinylated Donor Enhances Homology-Directed Repair in Combination with Cas9 Monoavidin in Mammalian Cells", 《THE CRISPR JOURNAL》 * |
| 刘志强: "《电子科技大学出版社》", 31 July 2017, 中国轻工业出版社 * |
| 李进等: "《现代发酵工程实验指导》", 31 May 2017, 电子科技大学出版社 * |
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