CN110004145A - A kind of sgRNA, knockout carrier, the knockout technique of KLF4 gene and its application - Google Patents
A kind of sgRNA, knockout carrier, the knockout technique of KLF4 gene and its application Download PDFInfo
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Abstract
The present invention discloses a kind of sgRNA, double-stranded DNA, carrier and cell.The invention also discloses a kind of knockout techniques of KLF4 gene.The IPEC-J2 cell line that the present invention utilizes CRISPR/Cas9 technology building KLF4 to knock out for the first time, can be used as the ideal cell model of pig disease research；Gene knockout is carried out using CRISPR/Cas9 technology, KLF4 gene can be more effectively knocked out than technological means such as gene silencing, interference, be more conducive to the functional study of KLF4 albumen；Gene sequencing detection shows that KLF4 gene order is knocked, and can cause the missing of KLF4 gene function, be the IPEC-J2 cell model of ideal KLF4 gene knockout.Knockout technique of the invention is easy, and target practice efficiency is high.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to the application technology of CRISPR/Cas9 is also related to chitterlings
Epithelial cell line (IPEC-J2) gene knockout carrier, gene knockout method and its application.
Background technique
CRISPR/Cas9 technology is derived from one of bacterium and Archimycetes and is situated between by sgRNA (small guide RNA)
The adaptive immune system led.Wherein sgRNA for identification, in conjunction with target spot DNA, it is combinable after Cas9 protein active is activated
The DNA of region downstream simultaneously cuts it, generates double-strand DNA cleavage, and induction is caused by non-homologous end joining repair mechanism
Frameshift mutation, to realize to the editor of target gene.CRISPR/Cas9 technology is because of its easy and efficient characteristic, in recent years
The important genetic approach for carrying out fixed point editor as eukaryotic gene obtains extensively in the mankind and animal-plant gene functional study field
General application.
Chitterlings epithelial cell (IPEC-J2) is isolated from the columnar epithelial cell in newborn piglet jejunum middle section, has research
The species specificity of porcine pathogen infection is the ideal external model for studying a variety of disease researches of pig, especially big in production enterotoxin
It is widely applied in enterobacteria and virus diarrhea resistant gene Function Identification research.
KLF4 is Kruppel sample transcription factor family member, and it is important to participate in regulating cell proliferation, differentiation, embryonic development etc.
Life process, and mediate innate immune response.There has been no the small of the knockout carrier of pig KLF4 gene and KLF4 gene knockout at present
Enterocyte model.
Summary of the invention
Goal of the invention: in order to overcome the shortcomings in the prior art, technical problem to be solved by the invention is to provide one
Kind sgRNA.
Also there is provided a kind of double-stranded DNAs for technical problems to be solved by the present invention.
Also there is provided a kind of carrier or cells for technical problems to be solved by the present invention.
There is provided a kind of knockout techniques of KLF4 gene for the last technical problems to be solved of the present invention.
Technical solution: in order to solve the above-mentioned technical problem, the technical scheme adopted by the invention is that: a kind of sgRNA, it is described
The base sequence of sgRNA is ACAGCCATGTCAGACTCGCC.
The content of present invention further includes a kind of double-stranded DNA, the upstream sequence of the double-stranded DNA are as follows:
SgRNA3F:
The downstream sequence of double-stranded DNA are as follows:
SgRNA3R:
The content of present invention also includes the carrier of the sgRNA or the double-stranded DNA, which is that one kind can practice shooting pig
The CRISPR/Cas9 knockout carrier of KLF4 gene.
The content of present invention also includes the cell of the carrier.
The content of present invention also includes a kind of knockout technique of KLF4 gene, comprising the following steps:
1) synthesis of target position point design and sgRNA sequence: the end of the area CDs 5 ' is found out according to pig KLF4 gene and carries out knockout target position
Point design sgRNA go-ahead sequence；
2) building of targeting vector: annealing the sgRNA go-ahead sequence of design to form double chain DNA sequence, then with carrier
It is connected to obtain connection product, connection product importing Escherichia coli is obtained into bacterium colony, bacterium colony extracts plasmid and obtains knockout carrier, root
According to knockout carrier and sgRNA sequence design PCR primer, positive practice shooting is obtained to the further sequence verification of the product of bacterium colony PCR and is carried
Body；
3) cell transfecting: by positive targeting vector be added in logarithmic growth phase IPEC-J2 cell suspension in into
Row electricity turns to obtain cell liquid；
4) knockout of KLF4 gene: puromycin is subjected to drug screening to cell liquid, the monoclonal cell of acquisition is turned
It moves and expands culture, when cell covers with, isolate part cell and extract its genomic DNA, according to KLF4 gene complete sequence,
The primer that target site nearby designs high specific is knocked out, PCR amplification then is carried out to the genomic DNA of extraction and obtains PCR product,
Product is verified.
Wherein, the PCR primer in the step 2) are as follows:
F3:
R3:
Wherein, the response procedures of bacterium colony PCR are 95 DEG C of 10min in the step 2)；95 DEG C of 35sec, 60 DEG C of 35sec, 72
DEG C 35sec, 35 circulations；72℃10min.
Wherein, IPEC-J2 cell concentration is 1~2 × 10 in the step 3)6A/ml.
Wherein, the primer sequence of the step 4) high specific are as follows:
F:
R:
Wherein, step 4) the PCR response procedures are 95 DEG C of 5min；95 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 10sec, 35
A circulation；72℃5min.
Knockout technique of the invention specifically includes the following steps:
1) by the sgRNA sequence of 5 ' end addition CACCG Add with 5 ' ends
The reverse complementary sequence of the sgRNA sequence of AAAC is added to anneal to form double-stranded DNA；
2) by the knockout carrier pGK2.1 connection of double-stranded DNA and linearisation, connection product is obtained；
3) with primer:
F1:
R1:
F2:
R2:
F3:
R3:
Bacterium colony PCR screening is carried out to connection product, is expanded culture and plasmid extracts, obtain positive targeting vector；
4) positive targeting vector is transfected into IPEC-J2 cell, extracts cell genomic dna, set near target site knocking out
The primer of high specific is counted, PCR amplification is carried out；
5)CruiserTMThe screening of digestion PCR product obtains 1 sgRNA that can efficiently knock out KLF4 gene；
6) PCR product sequence verification obtains the IPEC-J2 cell of KLF4 gene knockout.
The present invention knocks out KLF4 gene order in IPEC-J2 cell line genome using CRISPR/Cas9 technology, foundation
The IPEC-J2 cell of KLF4 gene knockout can be the mechanism of action deeply disclosed KLF4 gene in disease resistance and its resist in pig
Application in sick breeding provides more direct effective research model.
The utility model has the advantages that the present invention compared with the existing technology, has the advantage that
1, the IPEC-J2 cell line knocked out for the first time using CRISPR/Cas9 technology building KLF4, can be used as pig disease research
Ideal cell model；
2, gene knockout is carried out using CRISPR/Cas9 technology, it can be more effectively than technological means such as gene silencing, interference
KLF4 gene is knocked out, the functional study of KLF4 albumen is more conducive to；
3, gene sequencing detection shows that KLF4 gene order is knocked, and can cause the missing of KLF4 gene function, be more
The IPEC-J2 cell model of ideal KLF4 gene knockout.
4, knockout technique of the invention is easy, and provided sgRNA sequence can realize that high efficiency knocks out to target gene.
Detailed description of the invention
Fig. 1 is bacterium colony PCR detected through gel electrophoresis figure；
Fig. 2 is colony PCR product sequencing peak figure: Fig. 2A is that sgRNA1 corresponds to targeting vector sequencing peak figure；Fig. 2 B is
SgRNA2 corresponds to targeting vector sequencing peak figure；Fig. 2 C is that sgRNA3 corresponds to targeting vector sequencing peak figure；
Fig. 3 is to knock out target site PCR amplification detected through gel electrophoresis figure；
Fig. 4 is CruiserTMKLF4 Knockout cells DNAPCR product gel electrophoresis detection figure is screened in digestion；
Fig. 5 is CruiserTMDigestion screens KLF4 Knockout cells DNAPCR product and peak figure is sequenced；
Fig. 6 is that KLF4 Knockout cells DNA sequence dna and KLF4 gene do not knock out cell DNA sequence comparative analysis result.
Specific embodiment
The present invention will be further explained with reference to the accompanying drawing.
Embodiment 1
1. the synthesis of target position point design and sgRNA sequence:
The transcript CDs sequence of pig KLF4 gene (Gene ID:595111) is obtained according to ncbi database, and finds out CDs
The end of area 5 ' carries out knocking out target position point design, devises three using CRISPR Design (http://crispr.mit.edu/)
SgRNA go-ahead sequence:
In three sgRNA go-ahead sequence sgRNA1:SgRNA2:SgRNA3:Its 5 '
End addition CACCG, forms normal chain sgRNA DNA sequence dna；The reverse complementary sequence of simultaneously synthesizing normal chain sgRNA DNA sequence dna:
SgRNAl ':SgRNA2 ':SgRNA3 ':At 5 ' ends
AAAC is added, minus strand sgRNA DNA sequence dna is formed.
Three pairs of sgRNA sequences are as follows:
SgRNA1F:
SgRNA1R:
SgRNA2F:
SgRNA2R:
SgRNA3F:
SgRNA3R:
2. the building of targeting vector:
(1) the above positive and negative chain sgRNA sequence is subjected to annealing and forms double chain DNA sequence, reaction system is as follows:
Above-mentioned reaction system brief centrifugation is placed in 95 DEG C of incubations 3min, natural cooling 20min.
(2) linearization for enzyme restriction is carried out to knockout carrier pGK2.1 using BbsI enzyme, reaction system is as follows:
37 DEG C of incubation 4h, the carrier pGK2.1 of kits recycling linearisation.
(3) the carrier pGK2.1 of the double chain DNA sequence and linearisation that take above-mentioned annealing to be formed is attached, and reaction system is such as
Under:
After above-mentioned system brief centrifugation, 16 DEG C of incubation 30min obtain connection product.
(4) DH5 α competence is added in the above connection product of 10 μ l, mixes gently, is placed in and is incubated for 30min on ice；42 DEG C of heat
Swash 60sec, takes out set 2~3min of cooled on ice rapidly；Xiang Guanzhong 800 μ l nonreactive SOC fluid nutrient mediums of addition, shaking table (37 DEG C/
160rpm) cultivate 45min；4500rpm is centrifuged 5min, discards 600 μ l supernatants, precipitating is resuspended in remaining 200 μ l supernatant,
It is spread evenly across on the culture plate containing kalamycin resistance, is inverted 37 DEG C and is incubated overnight.
According to knockout carrier pGK2.1 and three sgRNA sequence designs three to PCR primer:
F1:
R1:
F2:
R2:
F3:
R3:
The screening of bacterium colony PCR: picking white monoclonal colonies are placed in PCR pipe from the above culture plate, into PCR pipe
Add each 1 μ l of above-mentioned upstream and downstream primer, buffer (10X) 2 μ l, dNTP 2 μ l, 0.2 μ l of Taq enzyme, 13.8 μ l of distilled water.PCR
Response procedures are 95 DEG C of 10min；95 DEG C of 35sec, 60 DEG C of 35sec, 72 DEG C of 35sec, 35 circulations；72℃10min.
Fig. 1 is bacterium colony PCR detected through gel electrophoresis figure, wherein M is 100bp DNA Marker, and 1,2,3 be positive bacterium colony
PCR band.As seen from Figure 1: above method obtains positive targeting vector.
To the obtained further sequence verification of positive colony PCR product, as shown in Fig. 2, positive targeting vector gene order
It matches with pGK2.1 carrier and sgRNA sequence.Positive targeting vector is expanded culture using conventional method, carries out plasmid
It extracts, is used for subsequent experimental.
3. cell transfecting:
About the 2 × 10 of logarithmic growth phase will be in6A chitterlings epithelial cell (IPEC-J2) is placed in 15mL centrifuge tube,
1000rpm/5min centrifugation is abandoned supernatant and is transferred in 1.5mL centrifuge tube with 210 μ l DPBS suspension cells.By 5~8 μ g above-mentioned 3
Kind targeting vector is separately added into the above-mentioned 1.5mL centrifuge tube containing IPEC-J2 cell suspending liquid, is mixed gently, by mixed liquor
Turn pipette tips with Special electric to be transferred in electric shock cup, after liquid level protrusion, covers electric shock cup lid and as electroporation, 650V/30ms
It carries out electricity to turn, cell liquid is transferred in six orifice plate culture mediums later.
4.KLF4 the knockout of gene:
After electricity turns 72 hours, medicine sieve is carried out with the puromycin of 3 μ g/ml concentration, and extremely with limiting dilution assay diluting cells
In 10 piece of 96 orifice plate, in 37 DEG C of CO2In incubator after stationary culture 7 days, monoclonal growing state is observed, it will be single after about 14 days
Clone cell, which is transferred in 48 holes, expands culture.When cell covers with 48 hole, part cell is isolated, extracts reagent using DNA
Box extracts its genomic DNA.
The DNA extracted in 5.PCR amplification step 4:
According to KLF4 gene complete sequence, the primer that high specific is designed near target site, sequence are being knocked out are as follows:
F:
R:
Pcr amplification reaction system is as follows:
PCR response procedures are 95 DEG C of 5min；95 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 10sec, 35 circulations；72℃
5min, 95 DEG C of 3min obtain the PCR product that length is 326 bp.
Fig. 3 is to knock out target site PCR amplification detected through gel electrophoresis figure.Wherein, M is 100bp DNA Marker, and 1,2,3 are
PCR product band.As seen from Figure 3: Successful amplification can be carried out to aim sequence by knocking out the primer that target site nearby designs.
The screening and verifying of 6.KLF4 Knockout cells:
(1)CruiserTMKLF4 Knockout cells are screened in digestion
Use the Cruiser of identification mispairingTMEnzyme carries out digestion detection, configuration to the PCR product that above-mentioned length is 326 bp
Reaction system is as follows:
2 μ l 6 × Stop Buffer are added in 45 DEG C of 10 μ l reaction systems of reaction 20min, Xiang Shangshu, carry out agar later
Sugared detected through gel electrophoresis.Fig. 4 is CruiserTMKLF4 Knockout cells are screened in digestion as a result, wherein M is represented
100bpDNAMarker；1-1,1-2 represent the cell of transfection sgRNA1 targeting vector；2-1,2-2 represent transfection sgRNA2 and practice shooting
The cell of carrier；3-1,3-2 represent the cell of transfection sgRNA3 targeting vector.As shown in Figure 4, sgRNA1 and sgRNA2 cannot be right
KLF4 gene is effectively knocked out, and sgRNA3 can effectively knock out KLF4 gene.Target practice efficiency analysis, knot are carried out according to gray value
Fruit shows, KLF4 gene targeting efficiency is respectively 54.8%, 56.1% in cell corresponding to 3-1 and 3-2.
(2) KLF4 Knockout cells DNA PCR product sequence verification:
To CruiserTMThe KLF4 Knockout cells DNAPCR product that digestion screens carries out sequence verification, such as Fig. 5
It is sequenced shown in peak figure, determines that the cell of transfection sgRNA3 targeting vector is KLF4 Knockout cells.It is thin to KLF4 gene knockout
Born of the same parents DNA send sequencing after being TA clone, does not knock out cell DNA sequence with KLF4 gene and is compared, the cell DNA sequence knocked out
Column sport the missing (Fig. 6) of 116bp and 137bp.
Fig. 6 is that TA cloning and sequencing analyzes KLF4 gene knockout sequence.Wherein, the first behavior KLF4 gene does not knock out
Sequence, the two or three behavior KLF4 gene knockout sequence.
The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Yangzhou University
<120>a kind of sgRNA, knockout carrier, the knockout technique of KLF4 gene and its application
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> sgRNA1 (Artificial Sequence)
<400> 1
acgttattcg gggcacctgc 20
<210> 2
<211> 19
<212> DNA
<213> sgRNA2(Artificial Sequence)
<400> 2
acaacactca cgttattcg 19
<210> 3
<211> 20
<212> DNA
<213> sgRNA3(Artificial Sequence)
<400> 3
acagccatgt cagactcgcc 20
<210> 4
<211> 24
<212> DNA
<213> F1(Artificial Sequence)
<400> 4
catatgctta ccgtaacttg aaag 24
<210> 5
<211> 21
<212> DNA
<213> R1(Artificial Sequence)
<400> 5
gcaggtgccc cgaataacgt c 21
<210> 6
<211> 24
<212> DNA
<213> F2(Artificial Sequence)
<400> 6
catatgctta ccgtaacttg aaag 24
<210> 7
<211> 19
<212> DNA
<213> R2(Artificial Sequence)
<400> 7
cgaataacgt gagtgttgt 19
<210> 8
<211> 24
<212> DNA
<213> F3(Artificial Sequence)
<400> 8
catatgctta ccgtaacttg aaag 24
<210> 9
<211> 20
<212> DNA
<213> R3(Artificial Sequence)
<400> 9
ggcgagtctg acatggctgt 20
<210> 10
<211> 25
<212> DNA
<213> sgRNA1F(Artificial Sequence)
<400> 10
caccgacgtt attcggggca cctgc 25
<210> 11
<211> 25
<212> DNA
<213> sgRNA1R(Artificial Sequence)
<400> 11
aaacgcaggt gccccgaata acgtc 25
<210> 12
<211> 24
<212> DNA
<213> sgRNA2F(Artificial Sequence)
<400> 12
caccgacaac actcacgtta ttcg 24
<210> 13
<211> 23
<212> DNA
<213> sgRNA2R(Artificial Sequence)
<400> 13
aaaccgaata acgtgagtgt tgt 23
<210> 14
<211> 25
<212> DNA
<213> sgRNA3F(Artificial Sequence)
<400> 14
caccgacagc catgtcagac tcgcc 25
<210> 15
<211> 24
<212> DNA
<213> sgRNA3R(Artificial Sequence)
<400> 15
aaacggcgag tctgacatgg ctgt 24
<210> 16
<211> 20
<212> DNA
<213> F(Artificial Sequence)
<400> 16
ggacttcgga ggacctctga 20
<210> 17
<211> 20
<212> DNA
<213> R(Artificial Sequence)
<400> 17
ccacccacag aagcccacta 20
Claims (10)
1. a kind of sgRNA, which is characterized in that the base sequence of the sgRNA is ACAGCCATGTCAGACTCGCC.
2. a kind of double-stranded DNA, which is characterized in that the upstream sequence of the double-stranded DNA are as follows:
SgRNA3F:5 '-CACCGACAGCCATGTCAGACTCGCC-3 ',
The downstream sequence of double-stranded DNA are as follows:
sgRNA3R: 5’-AAACGGCGAGTCTGACATGGCTGT-3’。
3. the carrier comprising sgRNA described in claim 1 or double-stranded DNA as claimed in claim 2.
4. including the cell of carrier as claimed in claim 3.
5. a kind of knockout technique of KLF4 gene, which comprises the following steps:
The synthesis of target position point design and sgRNA sequence: the end of the area CDs 5 ' is found out according to pig KLF4 gene and carries out knocking out target position point design
SgRNA go-ahead sequence；
The building of targeting vector: it anneals the sgRNA go-ahead sequence of design to form double chain DNA sequence, then be connected with carrier
Connection product is obtained, connection product importing Escherichia coli are obtained into bacterium colony, bacterium colony extracts plasmid and obtains knockout carrier, according to knockout
Carrier and sgRNA sequence design PCR primer obtain positive targeting vector to the further sequence verification of the product of bacterium colony PCR；
Cell transfecting: positive targeting vector is added in the suspension of the IPEC-J2 cell in logarithmic growth phase and carries out electricity turn
Obtain cell liquid；
The knockout of KLF4 gene: puromycin is subjected to drug screening to cell liquid, the monoclonal cell of acquisition is shifted and is expanded
Culture, when cell covers with, isolates part cell and extracts its genomic DNA, according to KLF4 gene complete sequence, is knocking out target
Location proximate designs the primer of high specific, then carries out PCR amplification to the genomic DNA of extraction and obtains PCR product, to product
It is verified.
6. the knockout technique of KLF4 gene according to claim 5, which is characterized in that the PCR primer in the step 2
Are as follows: F3:5 '-CATATGCTTACCGTAACTTGAAAG-3 '；
R3:5 '-GGCGAGTCTGACATGGCTGT-3 '.
7. the knockout technique of KLF4 gene according to claim 5, which is characterized in that bacterium colony PCR in the step 2
Response procedures are 95 DEG C of 10 min；95 DEG C of 35 sec, 60 DEG C of 35 sec, 72 DEG C of 35 sec, 35 circulations；72℃ 10
min。
8. the knockout technique of KLF4 gene according to claim 5, which is characterized in that IPEC-J2 is thin in the step 3)
Born of the same parents' concentration is 1 ~ 2 × 106A/ml.
9. the knockout technique of KLF4 gene according to claim 5, which is characterized in that the step 4) high specific draws
Object sequence are as follows:
F: 5'-GGACTTCGGAGGACCTCTGA-3'；
R: 5’-CCACCCACAGAAGCCCACTA-3’。
10. the knockout technique of KLF4 gene according to claim 5, which is characterized in that the step 4) PCR response procedures
For 95 DEG C of 5 min；95 DEG C of 10 sec, 60 DEG C of 10 sec, 72 DEG C of 10 sec, 35 circulations；72℃ 5 min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112011539A (en) * | 2020-08-26 | 2020-12-01 | 扬州大学 | Cell line for targeted knockout of porcine GDPD2 gene based on CRISPR-Cas9 technology and construction method thereof |
| CN112553200A (en) * | 2020-12-07 | 2021-03-26 | 扬州大学 | Preparation method and application of targeting vector for knocking out pig UGT2C1 gene |
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