CN110331169A - It is a kind of efficiently quickly in screening-gene regulatory region functional site method and application - Google Patents
It is a kind of efficiently quickly in screening-gene regulatory region functional site method and application Download PDFInfo
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Abstract
The invention discloses the methods and application of functional site in a kind of efficiently quick screening-gene non-coding regulatory region.The present invention combines Cre/lox and CRISPR/Cas9 technology, and external structure exomutation is introduced into genome, achievees the effect that be mutated in situ, to carry out the functional site of gene non-coding region (such as enhancer) efficient and quickly screening.By CRISPR/Cas9 technology, regulatory region introduces point mutation in genome, although can achieve the effect being mutated in situ, the heavy workload of its later period screening, in addition the design for being not appropriate for sgRNA in some mutational sites, is limited by the region of PAM sequence.The present invention breaches above-mentioned limitation, can carry out point mutation in any site in gene regulatory region.In addition this technology can introduce the excision of particular sequence in the mutation and gene regulatory region in two or more sites simultaneously, greatly reduce the difficulty of gene non-coding regulatory regional study.
Description
Technical field
The invention belongs to gene transcription level study on regulation fields, more particularly, to a kind of efficiently quick screening-gene
The method and application of functional site in regulatory region.
Background technique
It is all left in several kilobase the DNA length of functional area (such as enhancer) in the research of gene regulatory sequence
It is right, it is sometimes desirable to find out exact functional site, that is, focus on several bases.In order to find out functional site, need in genome
Mutation is introduced, to detect the importance in the site.
Traditional method is that the DNA fragmentation in the region is cloned into luciferase carrier, passes through luciferase signal
Change to react the importance in mutational site, but this method only detects in vitro, and the effect of abrupt climatic change not up in situ
Fruit.
Currently, existing research report introduces the mutation of specific site by CRISPR/Cas9 technology to study enhancer
(enhancer) functional site, but this technology have the defects that it is certain: the region of 1. NGG PAM sequences is restricted, i.e.,
The sequence in some sites is not suitable for being used as PAM sequence, can not just design sgRNA and targets the site and is mutated.2.
There is missing the target property in CRISPR/Cas9 technology.3. CRISPR/Cas9 technique successful rate is low, and only 1% or so.4. because of it
Success rate is low, and the later period needs a large amount of screening operation.These problems all considerably increase the difficulty of gene regulatory region research.
Summary of the invention
It is a kind of efficiently quickly in screening-gene regulatory region the purpose of the present invention is aiming at the shortcomings in the prior art, providing
The method of functional site.
The present invention combines Cre/lox and CRISPR/Cas9 technology, and exomutation is introduced into genome, reaches former
The effect that position is mutated, to efficiently and quickly sieve to the functional site of gene non-coding region (such as enhancer)
Choosing.By introducing resistant gene so as to carry out drug screening, it can substantially reduce and point mutation is introduced by CRISPR/Cas9 merely
Screening workload, while the region for also breaching NGG PAM sequence is restricted, can be (i.e. lox in gene regulatory region
Between point) arbitrarily site progress point mutation.In addition this technology can introduce mutation and the gene tune in two or more sites simultaneously
The excision for controlling particular sequence in region greatly reduces the difficulty of gene regulatory region research.
Specifically, the present invention is achieved by the following technical solutions:
(1) clone of gene regulatory sequence upstream region and downstream area
Gene regulatory sequence (by taking enhancer enhancer as an example, the same below), by round pcr by the upstream and downstream of enhancer
The region (such as Fig. 1) of region about 0.3-1kb clones respectively to be come, the plasmid construction for after.
(2) building (step a-c) (the plasmid tool of plasmid P, plasmid B, plasmid-lox-enh-wt, plasmid lox-enh-mut
Body structure is as shown in Figure 2)
A) site lox (lox71 and lox2272) is respectively added to puromycin/blasticidin-TK fusion first
With unmutated both ends enhancer (enh-wt)；
The puromycin/blasticidin-TK fusion includes puromycin/blasticidin(puromycin/kill
Piricularrin) resistant gene and thymidine kinase gene (TK suicide gene).Puromycin/blasticidin resistant gene is available
It is screened (i.e. positive-selecting) in corresponding resistant cell, TK gene can be used for reversely screening.
B) for plasmid P and plasmid B, it is also necessary to by the enhancer upstream region cloned in step (1) and downstream
Region is inserted respectively into the both ends for having connected the puromycin/blasticidin-TK fusion in the site lox, as
The homologous DNA fragment for the homologous recombination that CRISPR/Cas9 is mediated；
C) using the plasmid lox-enh-wt built as template, possible functional site is chosen, by point mutation PCR, obtains point
It is mutated the plasmid of enhancer, i.e. plasmid lox-enh-mut, which can contain one or more mutational sites.
(3) sgRNA-1 and sgRNA-2(Fig. 3 of targeting enhancer initiation site and end site is separately designed)；It will design
Good sgRNA is connected into px330 carrier (Cas9 and sgRNA expression vector), compiles for the subsequent CRISPR/Cas9 gene mediated
Volume.
(4) by the combination of CRISPR/Cas9 and Cre/lox technology come the point mutation in situ in mediated gene regulation region；
The step is the core technology of entire method, and mentality of designing is following (Fig. 4):
The homologous recombination mediated first by CRISPR/Cas9, by (the upstream and downstream area of enhancer of homologous fragment in plasmid
Domain) between DNA and genome in DNA between homologous fragment exchange, i.e., both ends are had to the puromycin/ in the site lox
Enhancer in the exogenous dna fragment replacement gene group of blasticidin-TK fusion, is knocked to obtain enhancer
Genome two sites lox and resistant gene are also inserted into genome with homologous recombination simultaneously.Enhancer is replaced thin
Born of the same parents have puromycin and blasticidin resistance simultaneously, can be by the way that drug puromycin and blasticidin, sieve is added
Choosing comes out the cell screening being replaced successfully.
Then by Cre/lox system, under the action of Cre enzyme, the unmutated property in plasmid between the site lox is enhanced
Son or saltant type enhancer go puromycin/blasticidin-TK in replacement gene group between the corresponding site lox to merge
Gene, the cell not being replaced successfully are substituted for because that, containing TK suicide gene, can be killed after drug GCV is added in genome
The cell of function can survive, the screening for following enhancer functional site.
Detailed process is as follows for the step (a-f) (Fig. 5):
A) each 2.5 μ g of plasmid P, plasmid B, px330-sgRNA-1 and px330-sgRNA-2 is taken, lipofectamine3000 is passed through
Transfection reagent box be transferred to completed in 6cm culture dish it is intracellular；
B) cell after transfecting is put into incubator, is cultivated 5-7 days；
C) puromycin(4 μ g/ml is added into culture medium) and blasticidin(10 μ g/ml), screening has puromycin
With the cell of blasticidin resistance；
D) culture by resisting cell with the concentration dilution in 0.5/hole into 96 orifice plates, for positive monoclonal cell strain；
E) design primer detects that (i.e. enhancer knocks out positive monoclonal cell strain, has simultaneously by PCR and sanger sequencing
Have the cell strain of puromycin and blasticidin resistance), and positive monoclonal is expanded and is cultivated, it is used for next step Cre/
The saltant type or the replacement of unmutated type enhancer that lox is mediated；
F) with lipofectamine3000 transfection reagent box by Cre plasmid respectively with plasmid lox-enh-wt or lox-enh-mut
Being transfected into the cell that filters out in step 5, (corresponding Cre plasmid can be bought from the website addgene, for the cell of different plant species
Select the plasmid of corresponding promoter driving expression).By culture in 4-5 days, drug GCV(5 μ g/ml is added) it carries out 5 days
Screening removes the cell containing TK gene.The genetic recombination mediated by Cre/lox, thus make in plasmid lox71 and
Unmutated property enhancer (enh-wt) or saltant type enhancer (enh-mut) between lox2272 are gone corresponding in replacement gene group
Two sites lox identify puromycin/blasticidin-TK fusion, and the cell that do not replace because containing TK from
It kills gene, can be killed after drug GCV is added, thus the cell strain and the enhancer containing mutational site that construct unmutated enhancer
Cell strain.Similarly, each mutational site can construct a corresponding cell strain.
(5) RNA of the cell strain of unmutated enhancer and the cell strain of different mutational site enhancers is extracted, it is fixed with fluorescence
Expression of the gene that amount PCR goes detection enhancer to be regulated and controled in different cell strains, the cell strain that gene expression amount is obviously lowered
Corresponding site is offunctional site, i.e., the expression that will lead to gene after the site mutation reduces.
Detailed description of the invention
Attached drawing 1 is regulating and controlling sequence in genome (enhancer) schematic diagram.
Attached drawing 2 is the related plasmids for needing to construct: plasmid P, plasmid B, plasmid-lox-enh-wt, plasmid lox-enh-mut
Schematic diagram.
Attached drawing 3 is sgRNA design site, and sgRNA-1 is located at enhancer starting point, and sgRNA-2 is located at enhancer terminating point.
Attached drawing 4 is that CRISPR/Cas9 and Cre/lox combine the mentality of designing being mutated in situ mediated.
Attached drawing 5 is that core procedure 4(CRISPR/Cas9 and Cre/lox combine the mutation in situ mediated) primary operational
Process.
Attached drawing 6 is the knockout of .PCR and sanger sequence verification enhancer enhancer-H.
Attached drawing 7 is that enhancer en-H knocks out and restore (being put back into genome by Cre/lox) to Ikzf1 expression
It influences.
Attached drawing 8 is the influence that different mutational sites are detected by qPCR to Ikzf1 gene expression.
Attached drawing 9 is to detect the site A and C by qPCR and western blot to be individually mutated with simultaneous mutation to Ikzf1 base
Because of the influence of expression.
The beneficial effect comprise that
(1) present invention combines Cre/lox and CRISPR/Cas9 technology, and exomutation is introduced into genome, reaches in situ
The effect of mutation can more accurately detect influence of the site to institute's controlling gene.
(2) present invention can substantially reduce by introducing resistant gene so as to carry out drug screening and pass through CRISPR/ merely
Cas9 introduces the workload of the screening of point mutation, while the region for also breaching NGG PAM sequence is restricted, can be in gene regulation
Any site carries out point mutation (i.e. between the site lox) in region, so as to gene non-coding regulatory region (such as enhancer
Deng) functional site carry out efficiently and quickly screening.
(3) present invention can introduce particular sequence in the mutation and gene regulatory region in two or more sites simultaneously
Excision, can study two or more sites and the function in terms of gene transcription regulation of certain segment DNA simultaneously, greatly reduce
The difficulty of gene regulatory region research.
Specific embodiment
Following example is only to further illustrate the present invention, and the invention is not limited in any way.
By taking the Ikzf1 gene of mouse as an example, it is that Ikzf1 gene expression must that enhancer enhancer-H, which is accredited out,
It needs, length about 4kb, on 11 chromosomes of mouse genome, Chr11:11740650-11744587.By upper
The method of telling has found two crucial functional sites on enhancer enhancer-H.
Specific research step is as follows:
(1) pass through round pcr for Ikzf1 enhancer enhancer-H upstream region 1kb (Chr11:11739626-
11740650) come with cloning for downstream area 1kb (Chr11:11744587-11745598)；
(2) (plasmid is specifically tied for the building (step a-c) of plasmid P, plasmid B, plasmid-lox-enh-wt, plasmid lox-enh-mut
Structure is as shown in Figure 2)
A) site lox (lox71 and lox2272) is respectively added to puromycin/blasticidin-TK fusion first
With unmutated both ends enhancer (enh-wt)；
B) for plasmid P and plasmid B, it is also necessary to by the enhancer upstream region 1kb cloned in step (1) and catchment
Domain 1kb is inserted respectively into the both ends for having connected the puromycin/blasticidin-TK fusion in the site lox；
C) using the plasmid lox-enh-wt built as template, possible functional site (site A ~ R) is chosen, point mutation is passed through
PCR obtains the plasmid of point mutation enhancer, i.e. plasmid lox-enh-mut, totally 18 plasmids containing single mutational site.It is (subsequent
The plasmid in two sites of simultaneous mutation can also be constructed)
(3) targeting enhancer initiation site and its sequence of the sgRNA-1 and sgRNA-2(of end site such as following table are separately designed)；
Designed sgRNA is connected into px330 carrier (Cas9 and sgRNA expression vector), is mediated for subsequent CRISPR/Cas9
Gene editing.
(4) by the combination of CRISPR/Cas9 and Cre/lox technology come the point mutation in situ in mediated gene regulation region；
Detailed process is as follows for the step (a-f):
A) each 2.5 μ g of plasmid P, plasmid B, px330-sgRNA-1 and px330-sgRNA-2 is taken, lipofectamine3000 is passed through
Transfection reagent box be transferred to completed in 6cm culture dish it is intracellular；
B) cell after transfecting is put into incubator, is cultivated 5-7 days；
C) puromycin(4 μ g/ml is added into culture medium) and blasticidin(10 μ g/ml), screening has puromycin
With the cell of blasticidin resistance；
D) culture by resisting cell with the concentration dilution in 0.5/hole into 96 orifice plates, for positive monoclonal cell strain；
E) design primer detects that (i.e. enhancer knocks out positive monoclonal cell strain, has simultaneously by PCR and sanger sequencing
Have the cell strain of puromycin and blasticidin resistance), and positive monoclonal is expanded and is cultivated, it is used for next step Cre/
The saltant type or the replacement of unmutated type enhancer that lox is mediated；
F) with lipofectamine3000 transfection reagent box by Cre plasmid respectively with plasmid lox-enh-wt or lox-enh-mut
Being transfected into the cell that filters out in step 5, (corresponding Cre plasmid can be bought from the website addgene, for the cell of different plant species
Select the plasmid of corresponding promoter driving expression).By culture in 4-5 days, drug GCV(5 μ g/ml is added) it carries out 5 days
Screening removes the cell containing TK gene.The genetic recombination mediated by Cre/lox, thus make in plasmid lox71 and
Unmutated property enhancer (enh-wt) or saltant type enhancer (enh-mut) between lox2272 are gone corresponding in replacement gene group
Two sites lox identify puromycin/blasticidin-TK fusion, and the cell that do not replace because containing TK from
It kills gene, can be killed after drug GCV is added, thus the cell strain and the enhancer containing mutational site that construct unmutated enhancer
Cell strain (A ~ R totally 18 plants of cell strains containing single mutational site).
(5) RNA of the cell strain of unmutated enhancer and the cell strain of different mutational site enhancers is extracted, it is fixed with fluorescence
Expression of the gene that amount PCR goes detection enhancer to be regulated and controled in different cell strains, the cell strain that gene expression amount is obviously lowered
Corresponding site is offunctional site, i.e., the expression that will lead to gene after the site mutation reduces.
After being knocked out enhancer-H by the above method in mouse RLM11 cell, and it is sequenced by PCR and sanger
Verify the effect knocked out, result such as Fig. 6, the binding site design primer of difference lox71 and lox2272, by DNA fragmentation 1,2
It clones to come respectively with 3, then carries out sequencing detection.The position of Fig. 6 (1) position PCR primer design, Fig. 6 (2) are the detection of PCR
As a result, can detecte enhancer-H(en-H in protogene group (wt)), it can't detect DNA fragmentation 1,2 and 3；And
Enhancer-H(en-H the genome detection after) knocking out can detecte less than enhancer-H to three binding site DNA pieces
Section 1,2 and 3.Fig. 6 (3) is the sequencing result of three binding site DNA fragmentations 1,2 and 3, further confirms that striking for enhancer-H
It removes and the insertion of the site lox and puromycin/blasticidin resistant gene.
We detect the expression of Ikzf1 gene from RNA and protein level, in figure 7 it is seen that working as
After enhancer-H is knocked out (enH-KO), Ikzf1 gene expression amount is substantially reduced, then will be unmutated by Cre/lox
Enhancer-H is put back into genome (lox-enH (wt)), and the expression quantity of Ikzf1 has the original level of recovery, while
It can be seen that not to the effect of enhancer in the both ends of the site lox insertion genome enhancer.
Next multiple plasmids containing different mutational sites (A ~ R) are constructed on the basis of our lox-enH-wt plasmids,
That is lox-enh-mut(A ~ R) by the method for above-mentioned described Cre/lox, we construct the more of the mutation containing different loci
A cell strain extracts the RNA of different cell strains, then detects the expression quantity of Ikzf1 gene in these cell strains (such as by qPCR
Fig. 8), after finding the site A and C site mutation, the expression of Ikzf1 is affected, decline is obvious, it was initially believed that the site A and C
Point is offunctional site, it may be possible to regulate and control the site of Ikzf1 gene transcription factor combination.As shown in figure 9, later we further through
Western further demonstrates qPCR's as a result, almost the same, while it is same by the above method can also to construct simultaneously the site A and C
When the cell strain that is mutated, the cell strain of the site A and C simultaneous mutation influences amount of its bigger expression decline on the expression quantity of Ikzf1
More.
Primer needed in the whole process is as listed by the following table 1,2 and 3:
1. QPCR primer of table
The PCR primer that 2. enhancer-H of table is verified after knocking out
The sgRNA primer sequence for the gene editing that 3. CRISPR/Cas9 of table is mediated
| sgRNA-1 | TTCTAGGCTGTTGAGGCCGT |
| sgRNA-2 | GGGAAAGGCCCTCATATCTC |
The above described is only a preferred embodiment of the present invention, be not intended to limit the present invention in any form, although this
Invention has been disclosed in a preferred embodiment above, and however, it is not intended to limit the invention, any person skilled in the art,
It does not depart within the scope of technical solution of the present invention, when the technology contents using the disclosure above make a little change or are modified to equivalent
The equivalent embodiment of variation, but without departing from the technical solutions of the present invention, according to the technical essence of the invention to the above reality
Any simple modification, equivalent change and modification made by example are applied, all of which are still within the scope of the technical scheme of the invention.
Claims (3)
1. it is a kind of efficiently quickly in screening-gene regulatory region functional site method, it is characterised in that: by Cre/lox and
CRISPR/Cas9 technology combines, and the mutation that external source constructs is introduced into genome, achievees the effect that be mutated in situ, while
Multidigit point simultaneous mutation can be carried out, to efficiently and quickly screen to the functional site of gene non-coding region.
2. the method for functional site, feature in a kind of efficiently quick screening-gene regulatory region according to claim 1
Be the following steps are included:
(1) clone of gene regulatory sequence upstream region and downstream area
Gene regulatory sequence (by taking enhancer enhancer as an example, the same below), by round pcr by the upstream and downstream of enhancer
The region (such as Fig. 1) of region about 0.3-1kb clones respectively to be come, the plasmid construction for after；
(2) (plasmid is specifically tied for the building (step a-c) of plasmid P, plasmid B, plasmid-lox-enh-wt, plasmid lox-enh-mut
Structure is as shown in Figure 2)
A) site lox (lox71 and lox2272) is respectively added to puromycin/blasticidin-TK fusion first
With unmutated both ends enhancer (enh-wt)；
The puromycin/blasticidin-TK fusion includes puromycin/blasticidin(puromycin/kill
Piricularrin) resistant gene and thymidine kinase gene (TK suicide gene)；
Puromycin/blasticidin resistant gene can be used for being screened (i.e. positive-selecting), TK to corresponding resistant cell
Gene can be used for reversely screening；
B) for plasmid P and plasmid B, it is also necessary to the enhancer upstream region and downstream area that will have been cloned in step (1)
The both ends for having connected the puromycin/blasticidin-TK fusion in the site lox are inserted respectively into, as CRISPR/
The homologous DNA fragment for the homologous recombination that Cas9 is mediated；
C) using the plasmid lox-enh-wt built as template, possible functional site is chosen, by point mutation PCR, obtains point
It is mutated the plasmid of enhancer, i.e. plasmid lox-enh-mut, which can contain one or more mutational sites；
(3) sgRNA-1 and sgRNA-2(Fig. 3 of targeting enhancer initiation site and end site is separately designed)；It will be designed
SgRNA is connected into px330 carrier (Cas9 and sgRNA expression vector), the gene editing mediated for subsequent CRISPR/Cas9；
(4) by the combination of CRISPR/Cas9 and Cre/lox technology come the point mutation in situ in mediated gene regulation region；
The step is the core technology of entire method, and mentality of designing is following (Fig. 4):
The homologous recombination mediated first by CRISPR/Cas9, by (the upstream and downstream area of enhancer of homologous fragment in plasmid
Domain) between DNA and genome in DNA between homologous fragment exchange, i.e., both ends are had to the puromycin/ in the site lox
Enhancer in the exogenous dna fragment replacement gene group of blasticidin-TK fusion, is knocked to obtain enhancer
Genome two sites lox and resistant gene are also inserted into genome with homologous recombination simultaneously；
The replaced cell of enhancer has puromycin and blasticidin resistance simultaneously, can be by the way that drug is added
Puromycin and blasticidin, screening come out the cell screening being replaced successfully；
Then by Cre/lox system, under the action of Cre enzyme, by plasmid between the site lox unmutated property enhancer or
Saltant type enhancer removes puromycin/blasticidin-TK fusion in replacement gene group between the corresponding site lox,
The cell not being replaced successfully is replaced successfully thin because that, containing TK suicide gene, can be killed after drug GCV is added in genome
Born of the same parents can survive, the screening for following enhancer functional site；
Detailed process is as follows for the step (a-f) (Fig. 5):
A) each 2.5 μ g of plasmid P, plasmid B, px330-sgRNA-1 and px330-sgRNA-2 is taken, lipofectamine3000 is passed through
Transfection reagent box be transferred to completed in 6cm culture dish it is intracellular；
B) cell after transfecting is put into incubator, is cultivated 5-7 days；
C) puromycin(4 μ g/ml is added into culture medium) and blasticidin(10 μ g/ml), screening has puromycin
With the cell of blasticidin resistance；
D) culture by resisting cell with the concentration dilution in 0.5/hole into 96 orifice plates, for positive monoclonal cell strain；
E) design primer detects that (i.e. enhancer knocks out positive monoclonal cell strain, has simultaneously by PCR and sanger sequencing
Have the cell strain of puromycin and blasticidin resistance), and positive monoclonal is expanded and is cultivated, it is used for next step Cre/
The saltant type or the replacement of unmutated type enhancer that lox is mediated；
F) with lipofectamine3000 transfection reagent box by Cre plasmid respectively with plasmid lox-enh-wt or lox-enh-mut
Being transfected into the cell that filters out in step 5, (corresponding Cre plasmid can be bought from the website addgene, for the cell of different plant species
Select the plasmid of corresponding promoter driving expression)；
By culture in 4-5 days, drug GCV(5 μ g/ml is added) screening that carries out 5 days, remove the cell containing TK gene；
The genetic recombination mediated by Cre/lox, to make the unmutated property enhancer in plasmid between lox71 and lox2272
(enh-wt) or saltant type enhancer (enh-mut) removes corresponding two sites lox identification puromycin/ in replacement gene group
Blasticidin-TK fusion, and the cell that do not replace can be killed after drug GCV is added because containing TK suicide gene
Extremely, to construct the cell strain of unmutated enhancer and the cell strain of the enhancer containing mutational site；
Similarly, each mutational site can construct a corresponding cell strain；
(5) RNA for extracting the cell strain of unmutated enhancer and the cell strain of different mutational site enhancers, uses fluorescent quantitation
Expression of the gene that PCR goes detection enhancer to be regulated and controled in different cell strains, the cell strain institute that gene expression amount is obviously lowered
Corresponding site is offunctional site, i.e., the expression that will lead to gene after the site mutation reduces.
3. the method for functional site, feature in a kind of efficiently quick screening-gene regulatory region according to claim 1
It is: is combined by Cre/lox and CRISPR/Cas9 technology, external structure exomutation is introduced into genome, reaches former
The effect that position is mutated, to efficiently and quickly sieve to the functional site of gene non-coding region (such as enhancer)
Choosing greatly reduces the difficulty of gene regulatory region research.
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| CN118072822A (en) * | 2024-02-04 | 2024-05-24 | 崖州湾国家实验室 | A pig functional mutation site gene chip based on interference transcription factor binding strength and its construction method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113584105A (en) * | 2021-08-04 | 2021-11-02 | 深圳瑞德林生物技术有限公司 | Method for preparing puromycin by enzyme method |
| CN113584105B (en) * | 2021-08-04 | 2023-10-20 | 深圳瑞德林生物技术有限公司 | Method for preparing puromycin by enzyme method |
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