CN108559760A - The method for establishing luciferase knock-in cell lines based on CRISPR targeted genomic modification technologies - Google Patents
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Abstract
The present invention relates to a kind of methods for establishing luciferase knock in cell lines based on CRISPR targeted genomic modification technologies, using CRISPR/Cas9 technologies in the genome SREBP1 genes 3 ' end united in situ enter T2A Luciferase reporter genes, the knock in cell lines of SREBP1 T2A Luciferase are established, and demonstrate the site-directed integration of foreign gene in the genome in this cell line.There are the transcription factor LXR α of activation to carry out transcriptional activation to SREBP1 T2A Luciferase cell lines SREBP1 using what is reported simultaneously, the results showed that the Luciferase expressions in SREBP1 T2A Luciferase cell lines accurately can delicately reflect that SREBP1 developed by molecule is horizontal in cell line.The foundation of the cell line, which will be helpful to SREBP1 gene functional research and screening, influences the small-molecule chemical drug of SREBP1 expression, and a kind of new experimental considerations and solution are provided for lipid metaboli and its correlative study.
Description
Technical field
The invention belongs to molecular biology fields, relate to the use of the target gene group insertion technology of CRISPR/Cas9 mediations
Establish cell line, and in particular to one kind establishing KI-T2A-Luciferase based on CRISPR/Cas9 targeted genomic modification technologies
The new method of cell line.
Background technology
CRISPR/Cas9 is bacterium and the immune defense system that archeobacteria long-term evolution is formed, which is to utilize
CRISPR-derivedRNA (crRNA) is combined to form compound by base pairing with trans-activating RNA, Cas9
Restriction endonuclease this compound guiding under pair with crRNA match sequence carry out fixed point cutting.Draw so having by engineer
The sgRNA (short guide RNA) for leading effect, can guide Cas9 to carry out fixed point cutting to host cell DNA, then pass through
Non-homologous end joining (non-homologous end joining, NHEJ) or homologous recombination (homologous
Recombination, HR) two kinds of mechanism for repairing approach are repaired, realize gene editing.
The expression regulation of detection gene is mainly by RT-PCR, Western Blot and by target gene promoter at present
The medium means of detection of expression carrier of reporter gene are cloned into realize.RT-PCR processes are cumbersome and unstable, Western
Blot antibody marks time-consuming costliness, these are all unfavorable for high flux screening；Since genome itself is there are epigenetics modification,
The method that promoter is cloned into the detection of expression carrier of reporter gene can not be simulated to the time of day of genome, therefore
It is difficult to accurately reflect expression conditions on genome.
With the continuous development for carrying out reporter gene technology this year, fluorescent protein report gene, luciferase reporter gene etc.
The method for having become common cell marking and tracer in basic research, since it has the advantages that convenient and efficient, safety is intuitive,
It is not only widely used in the molecular biology experiment for exploring structure molecular pathway, but also more and more extensive for screening drug institute structure
The cell line built.
Invention content
It is an object of the present invention to using CRISPR/Cas9 technologies, provide a kind of based on CRISPR/Cas9 target genes
Group modification technique establishes the new method of KI-T2A-Luciferase cell lines.
In order to realize that above-mentioned task, the present invention take following technical solution：
A kind of side establishing KI-T2A-Luciferase cell lines based on CRISPR/Cas9 targeted genomic modification technologies
Method, which is characterized in that follow these steps to implement：
1) corresponding sgRNA is designed and synthesized in the terminator codon downstream of target gene SREBP1, connected after annealing at room temperature
Enter pU6-sgRNA1.0, sgRNA is obtained after screening and expresses component, and detects its target practice efficiency；
2) the high sgRNA expression components of the target practice efficiency detected and Cas9 gene clonings are obtained to an expression vector
Obtain pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA；
3) the targeting vector pUC19/SREBP1-donor, targeting vector pUC19/ of structure targeting SREBP1 genes
The structure of SREBP1-donor is two parts, and first part is to be respectively provided with mutually homotactic upstream and downstream with broken site upstream and downstream
Homology arm；Second part be between upstream and downstream homology arm it is to be reorganized enter genome target site T2A-Luciferase-
CMV-eGFP-T2A-Neomycin-SV40pA DNA fragmentations；
4) pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA and pUC19/SREBP1-donor corotation HEK293 is thin
Born of the same parents are, after cell line stabilization, the G418 of 1.0mg/mL are added to screen 10 days, after cell line stabilization, then add the GCV of 10 μ g/mL
Screening 3 weeks after waiting for that cell line is stablized, carries out cloning, then carry out to the cell after cloning to cell through limiting dilution assay
PCR is identified and is sequenced, it was demonstrated that is carried correct recombination of the targeting vector of luciferase reporter gene at target site, is finally obtained
Obtain the HEK293-SREBP1-T2A-luciferase-KI cell lines of single stable；
5) clone and build the activating transcription factor LXR alpha expression carrier pUC19/CMV-LXR α of SREBP1 genes, the expression
Carrier pUC19/CMV-LXR α are used for the transcriptional activation experimental study of SREBP1 genes；
6) Luciferase expression variation in HEK293-SREBP1-T2A-luciferase-KI cell lines is verified whether may be used
Really to reflect relative expression quantity and the expression variation of endogenous SREBP1 genes；Use the transcription of constructed SREBP1 genes
Activity factor LXR alpha expression carrier pUC19/CMV-LXR α transfect HEK293-SREBP1-T2A-luciferase-KI cells respectively
System and wild type HEK293 cell lines, and in HEK293-SREBP1-T2A-luciferase-KI cell lines
The mRNA expressions of SREBP1 molecules are detected respectively in Luciferase activity and HEK293 cell lines；By right
SREBP1mRNA expressions and variation in knock-in cell lines in Luciferase expression activities and HEK293 cells
Compare, whether the Luciferase activity further verified in HEK293-SREBP1-T2A-luciferase-KI cell lines may be used
To accurately reflect relative expression's variation of SREBP1 molecules.
According to the present invention, the corresponding sgRNA of synthesis described in step 1) is the terminator codon downstream for SREBP1
The sgRNA of design, wherein：
SgRNA1 sequences are：GTCGAAGCTTTGAAGGCCGA；
SgRNA2 sequences are：GATCTTGACCCTAAGACCGG；
SgRNA3 sequences are：G TGGCCGATCGGGGCACTGC；
SgRNA4 sequences are：GCTTTCCCGGACTGCAAGCA.
Further, the HEK293 cell lines described in step 4) are human embryonic kidney cell line HEK293.
The present invention's establishes KI-T2A-Luciferase cell lines based on CRISPR/Cas9 targeted genomic modification technologies
New method, have the following advantages that：
Using CRISPR/Cas9 technologies in the genome SREBP1 genes 3 ' end united in situ enter T2A-Luciferase
Reporter gene, establishes the knock-in cell lines of SREBP1-T2A-Luciferase, and demonstrates external source base in this cell line
Because of site-directed integration in the genome.The transcription factor LXR α couple for having activation to SREBP1 reported are utilized simultaneously
SREBP1-T2A-Luciferase cell lines carry out transcriptional activation, the results showed that in SREBP1-T2A-Luciferase cell lines
Luciferase expressions accurately can delicately reflect in cell line that SREBP1 developed by molecule is horizontal.The cell line is built
The vertical SREBP1 gene functional research and screening of will be helpful to influences the small-molecule chemical drug of SREBP1 expression, be lipid metaboli and its
Correlative study provides a kind of new experimental considerations and solution.Meanwhile by target gene downstream be integrated into reporter gene come
The method of detection expression of target gene is also widely applied to the correlative study of various other genes.
Description of the drawings
Fig. 1 is the expression vector structural schematic diagram for carrying sgRNA and Cas9.
Fig. 2 is target practice Donor carrier structure schematic diagrames.
Fig. 3 is the structural schematic diagram of established KI-T2A-Luciferase cell lines.
Fig. 4 is to observe result figure after cell positive-negative selection is stablized under inverted fluorescence microscope, and wherein left figure is white light figure, right
Figure is fluorogram.
Fig. 5 is the Luciferase Activity determination figures each cloned after limiting dilution assay carries out cloning to cell.
Fig. 6 is to there is the active monoclonal cell PCR of Luciferase to detect electrophoretogram.
Fig. 7 is to wild type in the PCR product of No. 27 clones of gained after SREBP1-T2A-Luciferase Cell-cloneds
Size strip and integrated size strip carry out glue recycling sequencing result respectively.
Fig. 8 is activating transcription factor LXR alpha expression carrier pUC19/CMV-LXR α transfections HEK293-SREBP1-T2A-
The testing result of Luciferase after luciferase-KI cell lines.
After Fig. 9 is activating transcription factor LXR alpha expression carrier pUC19/CMV-LXR α transfected wild-type HEK293 cell lines,
The testing result of the mRNA expressions of SREBP1 molecules.
The present invention is described in further detail with reference to the accompanying drawings and examples.
Specific implementation mode
The present embodiment provides one kind and establishing KI-T2A- based on CRISPR/Cas9 targeted genomic modification technologies
The method of Luciferase cell lines generates double-strand notch (DSBs) using CRISPR/Cas9 systems in specific position,
A donor vehicle (Donor DNA) is added in Cas9-sgRNA components, the homologous of target site flank is carried on Donor DNA
Sequence, the reparation of DSBs can be carried out by template of donor dna, and then the genome that specific fragment is inserted into target gene is specific
On position.
Specifically implement according to the following steps：
1) corresponding sgRNA is designed and synthesized in the terminator codon downstream of target gene SREBP1, connected after annealing at room temperature
Enter pU6-sgRNA1.0, sgRNA is obtained after screening and expresses component, and detects its target practice efficiency；
2) the high sgRNA expression components of the target practice efficiency detected and Cas9 gene clonings are obtained to an expression vector
Obtain pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA；
3) the targeting vector pUC19/SREBP1-donor, targeting vector pUC19/ of structure targeting SREBP1 genes
SREBP1-donor structures are two parts, and first part is that be respectively provided with mutually homotactic upstream and downstream with broken site upstream and downstream same
Source arm；Second part be between upstream and downstream homology arm it is to be reorganized enter genome target site T2A-Luciferase-
CMV-eGFP-T2A-Neomycin-SV40pADNA segments；
4) pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA and pUC19/SREBP1-donor corotation HEK293 is thin
Born of the same parents are, after cell line stabilization, the G418 of 1.0mg/mL are added to screen 10 days, after cell line stabilization, then add the GCV of 10 μ g/mL
Screening 3 weeks or so after waiting for that cell line is stablized, carries out cloning, then to the cell after cloning to cell through limiting dilution assay
It carries out PCR identifications and is sequenced, it was demonstrated that carry correct recombination of the targeting vector of luciferase reporter gene at target site, most
The HEK293-SREBP1-T2A-luciferase-KI cell lines of single stable are obtained eventually；
5) clone and build the activating transcription factor LXR alpha expression carrier pUC19/CMV-LXR α of SREBP1 genes, the expression
Carrier pUC19/CMV-LXR α will be used for the transcriptional activation experimental study of SREBP1 genes；
6) Luciferase expression variation in HEK293-SREBP1-T2A-luciferase-KI cell lines is verified whether may be used
Really to reflect relative expression quantity and the expression variation of endogenous SREBP1 genes；Use the transcription of constructed SREBP1 genes
Activity factor LXR alpha expression carrier pUC19/CMV-LXR α transfect HEK293-SREBP1-T2A-luciferase-KI cells respectively
System and wild type HEK293 cell lines, and in HEK293-SREBP1-T2A-luciferase-KI cell lines
The mRNA expressions of SREBP1 molecules are detected respectively in Luciferase activity and HEK293 cell lines；By right
SREBP1mRNA expressions and variation in knock-in cell lines in Luciferase expression activities and HEK293 cells
Compare, whether the Luciferase activity further verified in HEK293-SREBP1-T2A-luciferase-KI cell lines may be used
To accurately reflect relative expression's variation of SREBP1 molecules.
In the present embodiment, the corresponding sgRNA of synthesis described in step 1) is the terminator codon downstream for SREBP1
The sgRNA of design, wherein：
SgRNA1 sequences are：GTCGAAGCTTTGAAGGCCGA；
SgRNA2 sequences are：GATCTTGACCCTAAGACCGG；
SgRNA3 sequences are：GTGGCCGATCGGGGCACTGC；
SgRNA4 sequences are：GCTTTCCCGGACTGCAAGCA.
The cell line of target practice Donor and Cas9-sgRNA the expression vector cotransfection is human embryonic kidney cell line
HEK293。
What the present embodiment provided establishes KI-T2A-Luciferase based on CRISPR/Cas9 targeted genomic modification technologies
The method of cell line is related to two crucial carrier structures, and one is the expression vector for expressing sgRNA and Cas9, and another kind is packet
The target practice Donor carriers of the homology arm containing upstream and downstream.
Wherein：
The expression vector of the expression sgRNA and Cas9 provided is that sgRNA is expressed component and Cas9 gene clonings to same
Constructed by one expression vector.
The target practice Donor carriers provided are by target practice broken site upstream 909bp and two sections of sequences of 887bp downstream
The upstream and downstream homology arm respectively as targeting vector is arranged, then itself and the T2A-Luciferase that applicant laboratory preserves are reported
Gene, positive screen element CMV-eGFP-T2A-Neomycin-SV40pA and negative screen element PGK-TK-T2A-mCherry-SV40pA
It is connected respectively in pU19 expression vectors constructed.
It is the specific embodiment that inventor provides below.
Embodiment 1：Target design, synthesis and the vector construction of the terminator codon downstream sgRNA of target gene SREBP1
(1) the terminator codon downstream of SREBP1 genes is chosen as targeting area (TSF), and length is about 1000bp；
(2) all NGG and its preceding 12 bit base are found out in the areas TSF and carries out Blast in NCBI, filtered out complete with target sequence
The sequence (if without satisfactory NGG, reversely searching CCN) of full matching and unique match, reduces potential site of missing the target；
The present embodiment devises 4 sgRNA, wherein：
SgRNA1 sequences are：GTCGAAGCTTTGAAGGCCGA；
SgRNA2 sequences are：GATCTTGACCCTAAGACCGG；
SgRNA3 sequences are：GTGGCCGATCGGGGCACTGC；
SgRNA4 sequences are：GCTTTCCCGGACTGCAAGCA.
5 ' positive oligonucleotides is obtained plus ACCG at it respectively, obtain its complementary strand, and 5 ' obtained plus AAAC at it
To reverse oligonucleotide.It will be connected into pU6-sgRNA1.0 after the forward and reverse oligonucleotides annealing at room temperature of synthesis, obtain sgRNA
Express component.
Embodiment 2：Express the vector construction of sgRNA components and Cas9 genes
(1) after being detected by T7E1, the high sgRNA expression vectors of target practice efficiency are filtered out；
(2) after the expression vector of the high sgRNA expression vectors of target practice efficiency and Cas9 being used I digestion of Kpn I and Spe respectively,
After the agarose gel electrophoresis recycling that mass concentration is 1%, obtained segment is connect with carrier, is reflected by digestion and sequencing
Surely positive colony is obtained.It is pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA (such as Fig. 1 institutes by the clone designation of acquisition
Show).
Embodiment 3：The structure of the targeting vector pUC19/SREBP1-donor of structure targeting SREBP1 genes
Constructed targeting vector includes target practice broken site upstream 909bp and two sections of sequence conducts of 887bp downstream
The upstream and downstream homology arm of targeting vector, wherein：
Upstream homology arm uses I for sequences of primer SREBP1up arm Cla
TATCGATGTCAGGCAGTGGTGGAGATG, I reverse sequences of SREBP1up arm Spe
GACTAGTGCTGGAAGTGACAGTGGTCC is obtained.
Downstream homology arm uses I for sequences of primer SREBP1down arm Sal
CGTCGACGGCCACAAGGTACACAACTTT, II reverse reverse sequences of SREBP1down arm Bgl
AAGATCTCTGTCCGTCCGTGTCCTCA is obtained.
T2A-Luciferase reporter genes, the positive screen element CMV-eGFP-T2A- that it is preserved with this laboratory again
Neomycin-SV40pA and negative screen element PGK-TK-T2A-mCherry-SV40pA are connected respectively to pU19 expression vectors, will obtain
The carrier obtained is named as pUC19/SREBP1-donor (as shown in Figure 2).
Embodiment 4：PCR sequencings are carried out to the cell line after cloning
The carrier that embodiment 2 and example 3 are obtained is with 4 μ g of mass ratio：8 μ g configurations, are transfected jointly using calcium phosphate method
HEK293 cells add the G418 of 1.0mg/mL to screen 10 days after cell line stabilization, after cell line stabilization, then add 10 μ g/mL
GCV screen 3 weeks, after waiting for that cell line is stablized, the expression (Fig. 4) of cell eGFP is observed with inverted fluorescence microscope, it
The Luciferase Activity determinations (Fig. 5) that cell is each cloned after limiting dilution assay carries out cloning afterwards, then to cloning
Cell afterwards carries out PCR identifications.PCR primer SREBP1integration detection for sequences
TGTGACCTGCTTCTTGTGGT, SREBP1integration detection reverse sequences
GGAGCGCAAAACCCAAGAAG (primer in the genome position as see arrows 17 in fig 3).PCR results show gained after cloning
The high active positive colonies of Luciferase have two band of wild type size strip and integrated size strip (such as Fig. 6 institutes respectively
Show).
Embodiment 5：The cell line after cloning is sequenced in TA clones
(1) two band of wild type size strip and integrated size strip of the clone No. 27 for respectively obtaining example 4
3 μ L of purified product connect with 0.5 μ LpGEM-T and convert 5 α competent cells of Escherichia coli DH；
(2) picking monoclonal primer T7 sequences TAATACGACTCACTATAGGG, primer SP6 sequences
ATTTAGGTGACACTATAG is sequenced, and sequencing result shows the targeting vector for carrying luciferase reporter gene at target site
Correct recombination, the final HEK293-SREBP1-T2A-luciferase-KI cell lines for obtaining single stable are (such as Fig. 7 institutes
Show).
Embodiment 5：Clone and build the activating transcription factor LXR alpha expression carriers of SREBP1 genes
Use I for sequence Cs CTCGAGATGTCCTTGTGGCTGGGG of primer LXR α CDS Xho, primer LXR α CDS Xba I
Reverse sequence Cs TCTAGATTCGTGCACATCCCAGAT obtains the activating transcription factor LXR α of SREBP1 genes, and builds and turn
The expression vector for recording activity factor LXR α obtains positive colony by digestion and sequencing identification.It is by the clone designation of acquisition
pUC19/CMV-LXRα。
Embodiment 6：Whether Luciferase expression variation can really reflect endogenous in the constructed cell line of verification
The relative expression quantity of SREBP1 genes and expression variation
Turned respectively using the activating transcription factor LXR alpha expression carrier pUC19/CMV-LXR α of constructed SREBP1 genes
The constructed knock-in cell lines of dye and wild type HEK293 cell lines, in constructed knock-in cell lines
The mRNA expressions of SREBP1 molecules detect (such as in Luciferase Activity determinations (as shown in Figure 8) and HEK293 cell lines
Shown in Fig. 9) display, compared with the control group, the significant enhancing of experimental group, it was confirmed that in constructed knock-in cell lines
Luciferase activity can accurately reflect relative expression's variations of SREBP1 molecules, the cell line established is named as
HEK293-SREBP1-T2A-luciferase-KI cell lines.
Claims (4)
1. a kind of method for establishing luciferase knock-in cell lines based on CRISPR targeted genomic modification technologies, feature
It is, follows these steps to implement：
1) corresponding sgRNA is designed and synthesized in the terminator codon downstream of target gene SREBP1, is connected into after annealing at room temperature
PU6-sgRNA1.0 obtains sgRNA and expresses component, and detects its target practice efficiency after screening；
2) it by the high sgRNA expression components of the target practice efficiency detected and Cas9 gene clonings a to expression vector, obtains
pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA；
3) the targeting vector pUC19/SREBP1-donor, targeting vector pUC19/SREBP1- of structure targeting SREBP1 genes
The structure of donor is two parts, and first part is to be respectively provided with mutually homotactic upstream and downstream homology arm with broken site upstream and downstream；
Second part be between upstream and downstream homology arm it is to be reorganized enter genome target site T2A-Luciferase-CMV-
EGFP-T2A-Neomycin-SV40pA DNA fragmentations；
4) by pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA and pUC19/SREBP1-donor corotation HEK293 cells
System adds the G418 of 1.0mg/mL to screen 10 days after cell line stabilization, after cell line stabilization, then adds the GCV sieves of 10 μ g/mL
It selects 3 weeks, after waiting for that cell line is stablized, cloning is carried out through limiting dilution assay to cell, then PCR is carried out to the cell after cloning
It identifies and is sequenced, it was demonstrated that correct recombination of the targeting vector of luciferase reporter gene at target site is carried, it is final to obtain
The HEK293-SREBP1-T2A-luciferase-KI cell lines of single stable；
5) clone and build the activating transcription factor LXR alpha expression carrier pUC19/CMV-LXR α of SREBP1 genes, the expression vector
PUC19/CMV-LXR α are used for the transcriptional activation experimental study of SREBP1 genes；
6) verify in HEK293-SREBP1-T2A-luciferase-KI cell lines whether Luciferase expression variation can be true
The relative expression quantity of real reflection endogenous SREBP1 genes and expression variation；Use the transcriptional activation of constructed SREBP1 genes
Factor LXR alpha expression carrier pUC19/CMV-LXR α transfect respectively HEK293-SREBP1-T2A-luciferase-KI cell lines and
Wild type HEK293 cell lines, and live to the Luciferase in HEK293-SREBP1-T2A-luciferase-KI cell lines
The mRNA expressions of SREBP1 molecules are detected respectively in property and HEK293 cell lines；By in knock-in cell lines
The comparison of Luciferase expression activities and SREBP1 mRNA expressions and variation in HEK293 cells, is further verified
Whether the Luciferase activity in HEK293-SREBP1-T2A-luciferase-KI cell lines can accurately reflect SREBP1
The relative expression of molecule changes.
2. the method as described in claim 1, which is characterized in that the corresponding sgRNA of synthesis described in step 1) be for
The sgRNA of the terminator codon downstream design of SREBP1, wherein：
SgRNA1 sequences are：GTCGAAGCTTTGAAGGCCGA；
SgRNA2 sequences are：GATCTTGACCCTAAGACCGG；
SgRNA3 sequences are：GTGGCCGATCGGGGCACTGC；
SgRNA4 sequences are：GCTTTCCCGGACTGCAAGCA.
3. the method as described in claim 1, which is characterized in that the HEK293 cell lines described in step 4) are human embryonic kidney cells
It is HEK293.
4. the method as described in claim 1, which is characterized in that the pCMV-Cas9-SV40pA-U6-sgRNAs-
SV40pA and pUC19/SREBP1-donor is with 4 μ g of mass ratio：8 μ g configurations.
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