CN105950622A - GS (glutamine synthetase) gene specific identification crRNA and application thereof - Google Patents
GS (glutamine synthetase) gene specific identification crRNA and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of cell and gene engineering, genetic modification and therapeutic recombinant protein industrial production, and particularly relates to GS (glutamine synthetase) gene specific identification crRNA and application thereof. DNA (deoxyribonucleic acid) sequences identified by the GS gene specific identification crRNA are selectively DNA sequences shown as one of sequences SEQ ID NO.1-SEQ ID NO.8. The GS gene specific identification crRNA and the application have the advantages that GS genes of diversified cells can be specifically identified by the GS gene specific identification crRNA, the GS gene specific identification crRNA is wide in applicability, and integral procedures can be implemented easily and efficiently; the target protein expression quantities of cells without the GS genes can be greatly increased on the basis of imported exogenous carriers as compared with wild host cells, and accordingly required-to-be-inputted labor, material resources and financial resources for screening protein expression cell strains can be reduced; the GS genes are knocked out, and accordingly the industrial production cost can be reduced; the cells without the GS are used as host target gene expression cells, other chemical substances can be omitted in procedures for producing the cells, and accordingly the production safety can be improved.
Description
Technical field
The invention belongs to cytogene engineering, genetic modification and therapeutic recombinant proteins field of industrialized production,
It is specifically related to crRNA and the application thereof of specific recognition GS gene.
Background technology
Mammalian cell is the main tool producing treatment complex proteins medicine at present, and wherein China
Hamster ovary cell (Chinese Hamster Ovary Cells, CHO) is the most frequently used host cell, at present
Have been widely used for commercial production.In protein expression system based on Chinese hamster ovary celI, dihydrofolate dehydrogenase
(Dihydrofolate Reductase, DHFR) and glutamine synthetase (Glutamine Synthetase, GS)
For two the most frequently used Select genes.Wherein GS system is because its ease for use and stability, is increasingly becoming and makes
By widest expression screening system.GS is the key enzyme (critical in L-glutaminate route of synthesis
Enzyme), its disappearance can cause cell self cannot synthesize L-glutamine, thus need to rely on the external world
The L-glutamine that environment provides could survive.
Traditional GS system is by the way of the inhibitor MSX adding GS during cell screening
Obtain foreign protein overexpression cell line.For in theory, the only cell of high expressed GS gene could be at height
The MSX of concentration survives under conditions of existing；After the transfection GS expression vector containing genes of interest, carrier does not has
Have and be incorporated into the cell of host cell gene group DNA or vector integration has arrived the cell of genome silent region,
All will be unable to survive under conditions of high concentration MSX exists.But, practical situation is, similar CHO-K1,
CHO-S, CHOK1SV these comprise the cell of normally functioning GS gene, without L-glutamine
Culture medium in can grow, certain some cell high concentration MSX exist under conditions of still can grow.
The foundation obtaining conclusions is, uses MSX to screen the clone obtained, has and do not have purpose egg the most at high proportion
White expression.This illustrates traditional Chinese hamster ovary celI containing normal GS gene expressive host as recombiant protein
This expression system, is a system the most rigorous.Therefore, the GS basal expression reducing host cell is the most non-
The most important, thus highlight the importance of the cell strain of GS genetic flaw.
The expression of recombinant proteins carrier containing GS Select gene is proceeded to, it will significantly carry in the cell of GS disappearance
The expression of high recombiant protein, reduces the workload needed for overexpression cell line screening.
The structure of GS deletion cells is the focus that over nearly 20 years, Chinese hamster ovary celI is engineered.All the time,
The technology relevant with GS, all because the problem of intellectual property is tied down.But, wherein a lot of technical protections arrive
2014 is the most expired.Therefore, a lot of technology for GS screening system are applied to business.Wherein than
More significant propelling factor is the fast development of gene editing technology, such as zinc finger endonucleases
(ZFN), mega-nucleases, TALENs and CRISPR.
CRISPR is the most comprehensive technology of function in gene editing technology, because his targeting mechanism is logical
Cross specific RNA sequence and be attached to, by complementary effect, the position that genome needs to carry out editing.
CRISPR is possibly realized by using RNA bunch to make to carry out polygenes editor simultaneously.Double-strand in cell is broken
Split (double strand break, DSB) and non-homologous end joining (non-homologous end can be passed through
Joining, NHEJ) mode be repaired, this frequency occurred of repairing is relatively low, and this reparation finally can
Cause gene to be sheared place's insertion of small fragment generation nucleic acid or disappearance, cause gene generation frameshit so that
Obtain genes of interest and lose function.
A kind of adaptive immunity that CRISPR system is antibacterial and archeobacteria is formed during long-term evolution is prevented
Imperial mechanism, can identify virus and the foreign DNA of the most reticent invasion.In recent years, due to technique for gene engineering
Advance by leaps and bounds, CRISPR has become as one of the most very powerful and exceedingly arrogant focus of scientific circles, is widely used in each
Interior and vitro system the hereditism of class body transforms, the structure of transgenic model animal, even field of gene.
One of them most important advantage of this system is that Cas9 albumen can under the guiding of multiple different crRNA simultaneously
Practice shooting multiple genomic locus.This advantage makes to be knocked out together by different genes to be possibly realized, the most simultaneously
The efficiency of gene editing is improve in degree.
CN 104651399 A discloses one and utilizes CRISPR/Cas system to realize base in Pig embryos cell
Because of the method knocked out, comprise the steps: to build Cas9 carrier for expression of eukaryon；Build gRNA expression vector；
Genes of interest target sequence is inserted in gRNA expression vector；By Cas9 carrier for expression of eukaryon and insert purpose
The insertion gRNA expression vector restricted enzyme linearisation of gene, then with linearizing plasmid as template
Carrying out in vitro transcription, in vitro transcription obtains the crRNA of mRNA and gRNA2# of hSpCas9 respectively,
The crRNA of mRNA and gRNA2# of hSpCas9 is by the kytoplasm of microinjection to pig IVF embryo at last
In, it is achieved knock out.This method effectively and low cost, is laid a good foundation for knocking out of other genes.
CN 102177235 A discloses a kind of I-CreI variant, one of two of which I-CreI monomer have to
Few two replacements, are wherein respectively arranged with one in two functional subdomains of LAGLIDADG Core domain,
Described subdomain lays respectively at 28 to 40 and 44 to 77 of I-CreI, and described variant can be cut
Cut the DNA target sequence from glutamine synthetase gene.This patent can only be for its of GS gene
In the 6th exon knock out, efficiency is low, and the process that knocks out is complicated.
Summary of the invention
For the deficiencies in the prior art and the demand of reality, the present invention provides specific recognition GS gene
CrRNA and application thereof, described crRNA can carry out genome with specific targeting GS gene to cell
Editor, the method can carry out mixing for multiple exons of GS gene and knock out, and efficiency is high, and security performance is high.
For reaching this purpose, the present invention by the following technical solutions:
First aspect, the present invention provides the crRNA of special target GS gene, and its targeting DNA sequence is selected from
DNA sequence shown in one of SEQ ID NO.1-SEQ ID NO.8, crRNA sequence is SEQ ID
The sequence of one of NO.9-SEQ ID NO.16.
Targeting DNA sequence (5 ' → 3 '):
SEQ ID NO.1:GCCATGTATATCTGGGTTGATGG
SEQ ID NO.2:GCCTGCAGGTGTGTATGGGGTGG
SEQ ID NO.3:GGCCTTCCAATGGCTTTCCTGGG
SEQ ID NO.4:GGTCCGTATTACTGTGGTGTGGG
SEQ ID NO.5:GTGGGCGCAGACAAAGCCTATGG
SEQ ID NO.6:GGTCAACATGTTCTTTCTAGTGG
SEQ ID NO.7:GGTCACAATTGGCAGAGGGGCGG
SEQ ID NO.8:GATGGCTTCTGTCACTGCAAAGG
CrRNA sequence (5 ' → 3 '):
SEQ ID NO.9:
GCCAUGUAUAUCUGGGUUGAGUUUUAGAGCUAUGCUGUUUUG
SEQ ID NO.10:
GCCUGCAGGUGUGUAUGGGGGUUUUAGAGCUAUGCUGUUUUG
SEQ ID NO.11:
GGCCUUCCAAUGGCUUUCCUGUUUUAGAGCUAUGCUGUUUUG
SEQ ID NO.12:
GGUCCGUAUUACUGUGGUGUGUUUUAGAGCUAUGCUGUUUUG
SEQ ID NO.13:
GUGGGCGCAGACAAAGCCUAGUUUUAGAGCUAUGCUGUUUUG
SEQ ID NO.14:
GGUCAACAUGUUCUUUCUAGGUUUUAGAGCUAUGCUGUUUUG
SEQ ID NO.15:
GGUCACAAUUGGCAGAGGGGGUUUUAGAGCUAUGCUGUUUUG
SEQ ID NO.16:
GAUGGCUUCUGUCACUGCAAGUUUUAGAGCUAUGCUGUUUUG
In the present invention, the crRNA designed according to the conserved sequence of the GS gene of various kinds of cell, it is applicable to many
Knocking out of the GS gene of kind cell, proceeds to host together with crRNA with Cas9 expression plasmid, tracrRNA thin
Born of the same parents, crRNA can knock out site at its corresponding GS of specific recognition, guides Cas9 albumen to specifying
Site is cut, in addition, and can be by being simultaneously directed a Cas9 albumen and multiple in cell
The mixing of the 2-7 exon that the mode of crRNA easily realizes GS gene knocks out, and improves efficiency.
Second aspect, the present invention provides a kind of CRISPR editing system for GS gene knockout, described table
The system of reaching includes RNA sequence as described in relation to the first aspect and Cas9 protein expressing plasmid.
The third aspect, the present invention provides a kind of host cell, and described host cell is use such as second aspect institute
The editing system gained stated.
Preferably, described host cell is beneficial to the expression of external source genes of interest.
Preferably, described destination protein be recombinant glycoprotein, recombinant hormone, restructuring enzyme, recombinant antibodies or
Any one in antibody fragment.
Preferably, described host cell is selected from mammalian cell.
Preferably, described mammalian cell is the derivative of CHO, NS0 or 293 cells and these three cell
Any one in cell.
Fourth aspect, the present invention provides a kind of utilization expression system as described in second aspect to knock out GS gene
Method, comprises the following steps:
(1) crRNA of selectively targeted GS gene is built；
(2) by crRNA and Cas9 protein expressing plasmid and the tracrRNA of step (1), mixing,
Proceed to host cell, to produce the cell of GS gene knockout；
(3) form proceeding to host cell in step (2) can be multiple: preferably, described side
Method is any one in electroporation transfection, liposome transfection, consideration convey dye.
5th aspect, the present invention provides the method as described in fourth aspect in preparation knocks out the cell of GS gene
Application.
Compared with prior art, there is advantages that
(1) crRNA of present invention design can the GS gene of specific identification various kinds of cell, the suitability
Extensively, transfection efficiency is high, and whole process operation is simple, efficiently；
(2) crRNA of the present invention can be by being simultaneously directed a Cas9 protein expressing plasmid and multiple
The mode of crRNA realizes one or several mixing in the 2-7 exon of GS gene and knocks out, and this is either
Knocking out in efficiency or degree easy and simple to handle is all that other technologies are incomparable；
(3) cell that the crRNA of the present invention knocks out is as host cell, uses the non-cell that knocks out to make with tradition
GS expression system for host is compared, after proceeding to foreign recombinant proteins expressing gene, it is possible to screening obtains high table
The probability of Dyclonine significantly increases, and the clone's quantity without protein expression and low expression is greatly reduced；
(4) cell that the crRNA of the present invention knocks out is as host cell, and it is improving high-expression clone ratio
Contribution so that the labour force that colony screening is required to be put into, high instrument expense (FACS, Clone Pix)
Etc. being greatly reduced, the cycle of high-expression clone simultaneously obtaining can be used for producing also is greatly shortened, from former
First 9～within 12 months, foreshorten to 4～5 months；
(5) present invention knocks out the cell of GS gene on the basis of importing foreign vector, can efficient table
Reach destination protein, by the raising of unit volume expression, reduce production scale, thus reduce industry
The beneficial effect of production cost, there is the chemical substance of risk of causing a disease, improves in production process without adding again
The safety produced.
Accompanying drawing explanation
Fig. 1 is Chinese hamster ovary cell GS gene structure figure.
Fig. 2 is the impact that L-Gln knocks out cell growth to GS.
Fig. 3 is growth and the graph of a relation of L-Gln in culture medium that wild-type cell and GS knock out cell.
Fig. 4 is the soda acid scattergram that GS knocks out cell glycoprotein identical with wild-type cell expression.
Fig. 5 is to knock out the application of the cell expression of recombinant proteins cell as host at 200L scale evaluation with GS.
Detailed description of the invention
By further illustrating the technological means and effect thereof that the present invention taked, below in conjunction with accompanying drawing and pass through
Detailed description of the invention further illustrates technical scheme, but the present invention is not limited to embodiment model
In enclosing.
Embodiment 1: for the structure of the CRISPR-Cas9 expression system of GS gene
The GS gene of Chinese hamster ovary cell comprises 7 exons, and wherein exon 2～7 codings are expressed
GS albumen (Fig. 1), the performance of normal function is played vital effect by it.For GS gene 2～7
Exon has separately designed crRNA sequence.These crRNA sequences and Cas9 protein is used to enable to
The GS albumen given expression to loses function.The exon2 of GS～following (the exon use of 7 exon partial sequences
Underscore " _ _ _ _ _ " labelling):
SEQ ID NO.17:
TGCCTCTTACGCAATTCCTGCAGGGGACCCCCTTCAGAGTAGATGTT
AATGAAATGACTTTTGTCTCTCCAGAGCACCTTCCACCATGGCCACCTCA GCAAGTTCCCACTTGAACAAAAACATCAAGCAAATGTACTTGTGCCTGCC CCAGGGTGAGAAAGTCCAAGCCATGTATATCTGGGTTGATGGTACTGGAG AAGGACTGCGCTGCAAAACCCGCACCCTGGACTGTGAGCCCAAGTGTGTA GAAGGTGAGCATGGGCAGGAGCAGGACATGTGCCTGGAAGTGGGCAAGC
AGCCTGAGATTTGACCTTCCTTCTGTTTTGTTTGCAAAGTCTTTCAAAAGC
AGGTCTCTTCAGGCCTCAGTCAGTCACCCGTAAGCTGCCGAGTAGTCTGG
AGG
SEQ ID NO.18:
ACTCAGTTACCAGCACCTACATGGTGGCTCACAACCATCTGTAACTC
CAATTTCAGGGGCTCCAACCCCCTCTTCTGCAGGCATACACTTGCACAGA
TATACATGCAAGTAAAACACCCCTACACACATAAAAATAAATACGTCTTC
TTAAAAGTTAATTTTCCATCTTTATTTGGCCCAGAGTTACCTGAGTGGAAT TTTGATGGCTCTAGTACCTTTCAGTCTGAGGGCTCCAACAGTGACATGTAT CTCAGCCCTGTTGCCATGTTTCGGGACCCCTTCCGCAGAGATCCCAACAA GCTGGTGTTCTGTGAAGTTTTCAAGTACAACCGGAAGCCTGCAGGTGTGT
ATGGGGTGGGCGTGAATGTCTTAAGAATCTAGGGATGGATGATCAGATGT
CCATCCTTCTACCCTGAACTTGCCTGCTGAAAAACAGTGTGGTCCGCCCCT
CCATGGTCCCTTTTATTGGTTGTATAAACAGTGTTGAATCTTCCATCTGT
SEQ ID NO.19:
TATAAAAAGTCTCTCAGCCTTTTCCTGCAAATGTACTATCATTGCTTC
TTCACAGTGGTTGGGCCTGAGTAGGTCCAGCCTATGATGACTTCAGCTGT
GTAAGAGTTGAGGACACTACTCCTTACAGCATGTTGATGCTTTATTCCTAGAGACCAATTTAAGGCACTCGTGTAAACGGATAATGGACATGGTGAGCAA CCAGCACCCCTGGTTTGGAATGGAACAGGAGTATACTCTGATGGGAACAG ATGGGCACCCTTTTGGTTGGCCTTCCAATGGCTTTCCTGGGCCCCAAGGTA
AGTTCCCCAGGTGAAATAAAAGCTTCCTCCCCATAAGTTCTTACTGTCCA
GAGACAGGAGCAGCTCCCAAATCAGCAAACAGACTGGCAGCTGAAAATA
AC
SEQ ID NO.20:
TAGTTCTACCAAGGCTACAGCAAAGAGGAAAGACCTAGCCTCCTACC
TGCAGGTGAAGACAGGATGTGCGAAAGCAAGTCTTAGGACCTTGTCATTT
TCTGGCTTTGGGGGGTTATGGACTCTGATTCTTCACTGATTGCTCTTGATT
CTCCTTCAGGTCCGTATTACTGTGGTGTGGGCGCAGACAAAGCCTATGGC AGGGATATCGTGGAGGCTCACTACCGCGCCTGCTTGTATGCTGGGGTCAA GATTACAGGAACAAATGCTGAGGTCATGCCTGCCCAGGTAAATGGCACTA
TT
SEQ ID NO.21:
CTGTTCCTTTTCCTCCCCTCTGAAGACTTGGCACATGGGGACTTTGGT
TAACAAGGGTGATGACTTAAAAGTGGTTCAGGGTAGAGGTAAGTAGAAC
AAGCTAGGAGCTTGAGTTGGCCTGAACAGTTAGTTGGCCTTATTCTAAAG GTCAACATGTTCTTTCTAGTGGGAATTCCAAATAGGACCCTGTGAAGGAA TCCGCATGGGAGATCATCTCTGGGTGGCCCGTTTCATCTTGCATCGAGTAT GTGAAGACTTTGGGGTAATAGCAACCTTTGACCCCAAGCCCATTCCTGGG AACTGGAATGGTGCAGGCTGCCATACCAACTTTAGCACCAAGGCCATGCG GGAGGAGAATGGTCTGAAGTAAGTAGCTTCCTCTGGAGCCATCTTTATTC
TCATGGGGTGGAAGGGCTTTGTGTTAGGGTTGGGAAAGTTGGACTTCTCA
CAAACTACATGCCATGCTCTTCGTGTTTGTCATAAGCCTATCGTTTTGTAC
C
SEQ ID NO.22:
GACTGGTCTGAAGCACTTGAGACATAGGTCACAAGGCAGACACAGC
CTGCATCAAGTATTTATTGGTTTCTTATGGAACTCATGCCTGCTCCTGCCC
TTGAAGGACAGGTTTCTAGTGACAAGGTCAGACCCTCACCTTTACTGCTT
CCACCAGGCACATCGAGGAGGCCATCGAGAAACTAAGCAAGCGGCACCG GTACCACATTCGAGCCTACGATCCCAAGGGGGGCCTGGACAATGCCCGTC GTCTGACTGGGTTCCACGAAACGTCCAACATCAACGACTTTTCTGCTGGT GTCGCCAATCGCAGTGCCAGCATCCGCATTCCCCGGACTGTCGGCCAGGA GAAGAAAGGTTACTTTGAAGACCGCCGCCCCTCTGCCAATTGTGACCCCT TTGCAGTGACAGAAGCCATCGTCCGCACATGCCTTCTCAATGAGACTGGC GACGAGCCCTTCCAATACAAAAACTAATTAGACTTTGAGTGATCTTGAGC
CTTTCCTAGTTCATCCCACCCCGCCCCAGCTGTCTCATTGTAACTCAAAGG
ATGGAATATCAAGGTCTTTTTATTCCTCGTGCCCAGTTAATCTTGCTTTTA
TTGGTCAGAATAGAGGAGTCAAGTTCTTAATCCCTATACACCCAACCCTC
ATTTCTTTTCTATTTAGCTTTCTAGTGGGGGTGGGAGGGGTAGGGGAAGG
GA
Following (table 1) for the crRNA sequence designed by above exon:
Table 1
Embodiment 2: CHO-K1 cell is carried out GS gene and knocks out and verify
By crRNA Yu the Cas9 protein expression vector that the designs CHO-K1 to taming through CD culture medium
Cell transfects, altogether design A～P totally 16 kinds of transfections or transfection combination.
CHO-K1 cell is purchased from ATCC, and cell is cultivated and used DMEM/F12 basal medium, adds 5%
Hyclone.Use CD CHO culture medium to enter suspension culture in the CHO-K1 cell after amplification, add
Glutamine and HT additive.
After cell adapted suspension culture, by crRNA Yu Cas9 protein expression vector transfection to cell.Turn
Add Puromycin after dye 48hr in cell culture medium, after cultivating 4 days, the cell selection of survival gone out,
Carry out sub-clone cultivation.After subcloned cells grows, cell is transferred to respectively containing L-Gln and without L-Gln
Culture medium cultivate.Cannot will grow in without the culture medium of L-Gln, in the culture medium containing L-Gln
In can pick out with the clone of normal growth, carry out amplification cultivation.
A～P totally 16 kinds of transfections or transfection combination are screened the GS obtained and knocked out cell and add up, data
Showing that carrying out 3 kinds to 4 kinds target spots knocks out jointly, it is the highest that it knocks out efficiency, the results are shown in Table 2.
Table 2
The clone of amplification cultivation is transferred to shaking flask cultivate.To can the cell of normal growth in shaking flask
Carry out Gln and remove checking, filter out under suspended state, grow, it is necessary to rely on the cell of Gln growth
(such as Fig. 2-3).By above method, screening obtains 20 strain GS gene delection cell strains altogether.
Embodiment 3:GS genetic flaw cells expressing antibody is tested
The GS KO1 picked out and GS KO2 cell are carried out antibody expression transfection experiment, wild type
CHO-K1 cell is as comparison host.By KJ015 by identical method transfection to GS KO1,
GS KO2, CHO-K1 cell.48hr after transfection, adds 25 μMs of MSX as screening pressure.Stand training
After supporting 2 weeks, clone grows, 25 clones the highest to the quantity and 24hr antibody expression amount growing clone
Contrasting, result is as shown in table 3.Clone's quantity variance that three grows is less, expression GS KO1
Expression obvious with the clone that GS KO2 is host is high.
Table 3
Expression preferably cell is carried out amplification cultivation, and collects expression product, with IEC as Key Quality
Index analysis expresses the quality of antibody.Result display GS knocks out cellular products quality and wild-type cell product
Quality can reach phase same level (table 4).
Table 4
Embodiment 4:GS genetic flaw cell expresses glycoprotein experiment
The GS KO1 picked out and GS KO2 cell are carried out P-glycoprotein expression transfection experiment, wild type
CHO-K1 cell is as comparison host.By KJ018 by identical method transfection to GS KO1,
GS KO2, CHO-K1 cell.48hr after transfection, adds 25 μMs of MSX as screening pressure.Stand training
After supporting 2 weeks, clone grows, and adds up the quantity and 24hr P-glycoprotein expression amount growing clone, knot
Fruit is as shown in table 5.Clone's quantity variance that three grows is less, and expression GS KO1 and GS KO2 is
The obvious expression of cell of host is high.
Table 5
Expression preferably cell is carried out amplification cultivation, and collects expression product, with IEF as Key Quality
Index analysis expresses the quality of glycoprotein.Result display GS knocks out cellular products quality and wild-type cell product
Quality can reach phase same level (Fig. 4).
Embodiment 5: the pilot scale culture (200L) of the antibody expressing cells with GS genetic flaw cell as host
Antibody overexpression cell line GS KO1A5 screening obtained carries out amplification cultivation, when carefully in shaking flask
When born of the same parents are in exponential phase, cell is seeded to 14L bioreactor, working volume 10L.Reacted
The cell of device amplification, is seeded to 200L reactor.After inoculation, cell density is 0.3 × 106About cells/mL,
It is 130L that reactor initiates working volume, sets parameters temperature 37 DEG C, pH 6.90-7.20, DO
20%-50%, every day, sterile sampling, detected glucose and lactate content.Cultivating the technique used is feed supplement
Criticize and cultivate.Incubation needs monitoring index have: temperature, pH, concentration of glucose, lactate concentration,
Morie osmolarity, marking protein concentration.
Cell was reactor growth 11 days, and antibody expression amount is 5.1g/L, and when terminating to cultivate, cell state is good
Good, vigor is 98% (Fig. 5).Result above shows, GS deficient cells can be trained in 200L scale
Support, and be able to maintain that good cell state, the high expressed of albumen and quality.According to this results presumption,
Host's GS deficient cells of this clone is applicable to the host cell of recombiant protein industrialized production cell.
Applicant states, the present invention illustrates the method detailed of the present invention by above-described embodiment, but the present invention
It is not limited to above-mentioned method detailed, does not i.e. mean that the present invention has to rely on above-mentioned method detailed and could implement.
Person of ordinary skill in the field is it will be clearly understood that any improvement in the present invention, each former to product of the present invention
The equivalence of material is replaced and the interpolation of auxiliary element, concrete way choice etc., all falls within the protection model of the present invention
Within the scope of enclosing and disclosing.
Claims (10)
1. the crRNA of special target GS gene, it is characterised in that described crRNA sequence is SEQ ID
The sequence of one of NO.9-SEQ ID NO.16, the DNA sequence of identification is selected from SEQ ID NO.1-SEQ
DNA sequence shown in one of ID NO.8.
2. the CRISPR gene editing system being used for knocking out GS gene, it is characterised in that described gene
Editing system include a kind of or at least two of crRNA sequence as claimed in claim 1 combination,
Cas9 protein expression vector and tracrRNA.
3. an expression of recombinant proteins host cell, it is characterised in that described host cell system is wanted by such as right
Ask the system described in 2 that this cell is carried out gene editing gained.
Host cell the most according to claim 3, it is characterised in that described host cell, its GS base
Because of afunction.
Host cell the most according to claim 3, it is characterised in that described host cell, it is possible to efficiently
Express the base sequence comprising external source destination protein；
Preferably, described destination protein be recombinant glycoprotein, recombinant hormone, restructuring enzyme, recombinant antibodies or
Any one in antibody fragment.
6. according to the host cell described in claim 4 or 5, it is characterised in that described host cell is selected from feeding
Breast zooblast；
Preferably, described mammalian cell is the derivative of CHO, NS0 or 293 cells and these three cell
Any one in cell.
7. utilizing the method that the gene editing system described in claim 2 knocks out GS gene, its feature exists
In, comprise the following steps:
(1) crRNA of selectively targeted GS gene is built；
(2) crRNA and tracrRNA and the Cas9 protein expression vector of step (1) is mixed, proceed to
In host cell, to produce the cell of GS gene merit disappearance；
(3) form proceeding to host cell in step (2) can be multiple:
Preferably, described method be electroporation transfection, liposome transfection, consideration convey dye in any one.
Method the most according to claim 7, it is characterised in that after step (2) described mixing, crRNA
It is 1:1 with the molar ratio of tracrRNA.
9. the application in preparation knocks out the cell of GS gene of the method described in claim 7 or 8.
The host cell the most according to claim 6 application in preparing recombiant protein；Preferably, described
Recombiant protein is clinical treatment medicine.
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| CN112646837A (en) * | 2019-10-12 | 2021-04-13 | 中国人民解放军总医院 | Recombinant human adiponectin expression vector, vector construction method and expression method |
| CN114317535A (en) * | 2021-12-02 | 2022-04-12 | 中国检验检疫科学研究院 | Gene deletion CHO cell line and preparation method and application thereof |
| CN114317535B (en) * | 2021-12-02 | 2024-01-05 | 中国检验检疫科学研究院 | Gene-deleted CHO cell line and preparation method and application thereof |
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