CN105950622B - The crRNA of specific recognition GS gene and its application - Google Patents
The crRNA of specific recognition GS gene and its application Download PDFInfo
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Abstract
The invention belongs to cytogene engineering, genetic modification and therapeutic recombinant proteins field of industrialized production, the DNA sequence dna of crRNA and its application more particularly to specific recognition GS gene, the crRNA identification are selected from DNA sequence dna shown in one of SEQ ID NO.1-SEQ ID NO.8.The crRNA that the present invention designs can specificity identification various kinds of cell GS gene, applicability is wide, and whole process is easy to operate, efficiently；The cell of GS gene is knocked out in the present invention on the basis of importing foreign vector, relative to wild-type host cells, the expression quantity of destination protein greatly improves, to reduce the human and material resources put into needed for protein expressing cells strain screening and financial resources, and industrial cost can be reduced by knocking out GS gene.Cell is knocked out as the destination gene expression cell of host with GS, and production process no longer needs to add other chemical substance, improves the safety of production.
Description
Technical field
The invention belongs to cytogene engineering, genetic modification and therapeutic recombinant proteins field of industrialized production, specifically
It is related to crRNA and its application of specific recognition GS gene.
Background technique
Mammalian cell is the main tool of current production treatment complex proteins drug, and wherein Chinese hamster ovum
Nest cell (Chinese Hamster Ovary Cells, CHO) is most common host cell, is had been widely used at present
Industrial production.In protein expression system based on Chinese hamster ovary celI, dihydrofolate dehydrogenase (Dihydrofolate Reductase,
DHFR) and glutamine synthelase (Glutamine Synthetase, GS) is most common two selections gene.Wherein GS system
System is increasingly becoming most popular expression screening system because of its ease for use and stability.GS is L-Glutamine synthesis
Key enzyme (critical enzyme) in approach, its missing, which will lead to cell itself, can not synthesize L-glutamine, from
And the L-glutamine for the external environment offer that needs to rely on could survive.
Traditional GS system is to obtain external source egg by way of adding the inhibitor MSX of GS during cell screening
White overexpression cell line.For theoretically, under the conditions of the cell of only high expression GS gene could be existing for the MSX of high concentration
Existence；Transfect the GS expression vector containing target gene after, carrier be not integrated into host cell gene group DNA cell or
Person's vector integration has arrived the cell of genome silent region, survives under the conditions of all will be unable to existing for the high concentration MSX.However, real
Border situation is that these include the cell of normally functioning GS gene by similar CHO-K1, CHO-S, CHOK1SV, is being free of L-
It can grow in the culture medium of glutamine, can still be grown under the conditions of certain some cell is existing for the high concentration MSX.?
Foundation to conclusions is that the clone screened using MSX has the very not expression of destination protein at high proportion.This says
Bright traditional Chinese hamster ovary celI containing normal GS gene is one not rigorous as this expression system of the expressive host of recombinant protein
System.Therefore, the GS basal expression of host cell is reduced with regard to extremely important, thus highlight the cell strain of GS gene defect
Importance.
The recombinant protein expression vector of the selection gene containing GS is transferred in the cell of GS missing, it will greatly improve recombination egg
White expression, workload needed for reducing overexpression cell line screening.
The building of GS deletion cells is the hot spot that Chinese hamster ovary celI is engineered in the past 20 years.It is all the time related to GS
Technology, all because the problem of intellectual property is tied down.But wherein many technical protections are expired to 2014.Therefore, very
The technology for being chiefly used in GS screening system is able to be applied to business.Wherein more significant propulsion factor is gene editing technology
Fast development, 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 by special
Property RNA sequence the position that genome is edited is integrated to by complementary effect.CRISPR is made by using RNA cluster
Polygenes editor must be carried out simultaneously to be possibly realized.Double-strand break (double strand break, DSB) in cell can lead to
The mode for crossing non-homologous end joining (non-homologous end joining, NHEJ) is repaired, what this reparation occurred
Frequency is lower, and this reparation finally will cause gene and be sheared nucleic acid occurs for place's small fragment insertion or missing, cause
Frameshit occurs for gene so that target gene loses function.
CRISPR system is a kind of adaptive immunity defense mechanism that bacterium and archeobacteria are formed during long-term evolution,
It can identify the virus and exogenous DNA of simultaneously silencing invasion.In recent years, advancing by leaps and bounds due to technique for gene engineering, CRISPR is
Become most very powerful and exceedingly arrogant one of the hot spot of scientific circles, be widely used in all kinds of internal and external systems science of heredity transformation,
The building or even field of gene of transgenosis model animal.One of them most important advantage of the system is that Cas9 albumen can
It practices shooting multiple genomic locus simultaneously under the guidance of multiple and different crRNA.This advantage makes different genes one simultaneously
It rises to knock out and be possibly realized, greatly enhance the efficiency of gene editing.
104651399 A of CN, which is disclosed, a kind of realizes gene knockout in Pig embryos cell using CRISPR/Cas system
Method, include the following steps: construct Cas9 carrier for expression of eukaryon；Construct gRNA expression vector；Target gene target sequence is inserted
Enter in gRNA expression vector；By Cas9 carrier for expression of eukaryon and the insertion gRNA expression vector of target gene is inserted with restricted
Restriction endonuclease linearisation, then be transcribed in vitro using the plasmid of linearisation as template, the mRNA for obtaining hSpCas9 respectively is transcribed in vitro
With the crRNA of gRNA2#, the crRNA of the mRNA of hSpCas9 and gRNA2# is finally passed through into the born of the same parents of microinjection to pig IVF embryo
In matter, realizes and knock out.This method is effective and at low cost, lays a good foundation for the knockout of other genes.
102177235 A of CN discloses a kind of I-CreI variant, and one of two of them I-CreI monomer has at least two
Replacement wherein respectively has one in the functional subdomain of the two of LAGLIDADG Core domain, and the subdomain distinguishes position
In 28 to 40 of I-CreI and 44 to 77, the variant can cut the DNA target from glutamine synthetase gene
Sequence.The patent can only be knocked out for wherein the 6th exon of GS gene, low efficiency, and it is complicated to knock out process.
Summary of the invention
In view of the deficiencies of the prior art and actual demand, the present invention provide specific recognition GS gene crRNA and its
Using the targeting GS gene that the crRNA can be specific carries out genome editor to cell, and this method can be directed to GS gene
Multiple exons carry out mixing knockout, high-efficient, security performance is high.
To achieve this purpose, the present invention adopts the following technical scheme:
In a first aspect, the present invention provides the crRNA of special target GS gene, targeting DNA sequence dna is selected from SEQ ID
DNA sequence dna shown in one of NO.1-SEQ ID NO.8, crRNA sequence are the sequence of one of SEQ ID NO.9-SEQ ID NO.16
Column.
It targets DNA sequence dna (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, according to the crRNA that the conserved sequence of the GS gene of various kinds of cell designs, it is applicable to various kinds of cell
The knockout of GS gene, crRNA are transferred to host cell together with Cas9 expression plasmid, tracrRNA, and crRNA can know in specificity
Not its corresponding GS knocks out site, and guidance Cas9 albumen is cut to specific bit point, in addition to this, can be by cell
In import the mode of a Cas9 albumen and multiple crRNA simultaneously and easily realize that the mixing of the 2-7 exon of GS gene knocks out,
It improves efficiency.
Second aspect, the present invention provide a kind of CRISPR editing system for GS gene knockout, the expression system packet
Include RNA sequence and Cas9 protein expressing plasmid as described in relation to the first aspect.
The third aspect, the present invention provide a kind of host cell, and the host cell is using the volume as described in second aspect
Obtained by the system of collecting.
Preferably, the host cell is conducive to the expression of external source target gene.
Preferably, the destination protein is recombinant glycoprotein, recombinant hormone, recombination enzyme, recombinant antibodies or antibody fragment
In any one.
Preferably, the host cell is selected from mammalian cell.
Preferably, the mammalian cell is the derivative of CHO, NS0 or 293 cells and these three cells
Any one in cell.
Fourth aspect, the present invention provide a kind of method that the expression system using as described in second aspect knocks out GS gene,
The following steps are included:
(1) crRNA of selectively targeted GS gene is constructed；
(2) by crRNA the and Cas9 protein expressing plasmid and tracrRNA of step (1), mixing is transferred to host cell
In, to produce the cell of GS gene knockout；
(3) form for being transferred to host cell in step (2) can be a variety of: preferably, the method is electroporation
Transfection, liposome transfection, any one in nuclear transfection.
5th aspect, the present invention provide method the answering in the cell that preparation knocks out GS gene as described in fourth aspect
With.
Compared with prior art, the invention has the following beneficial effects:
(1) the GS gene for the identification various kinds of cell that the crRNA that the present invention designs can be specific, applicability is wide, transfection effect
Rate is high, and whole process is easy to operate, efficiently；
(2) crRNA of the invention can be by way of importing a Cas9 protein expressing plasmid and multiple crRNA simultaneously
Realize that mixing one or several in the 2-7 exon of GS gene knocks out, this either knocks out efficiency or journey easy to operate
It is all that other technologies are incomparable on degree；
(3) cell that crRNA of the invention is knocked out uses non-knockout cell as host's as host cell with tradition
GS expression system is compared, and after being transferred to foreign recombinant proteins expressing gene, the probability that can screen to obtain high-expression clone substantially increases
Clone's quantity of height, no protein expression and low expression is greatly reduced；
(4) cell that crRNA of the invention is knocked out is used as host cell, in the contribution of raising high-expression clone ratio,
So that labour, the high instrument expense (FACS, Clone Pix) etc. that put into needed for colony screening are greatly reduced, together
When obtain can be used for period of the high-expression clone produced and also greatly shorten, foreshortened to 4~5 months from original 9~12 months；
(5) cell of GS gene is knocked out in the present invention on the basis of importing foreign vector, it can be with high efficient expression purpose egg
It is white, by the raising of unit volume expression quantity, production scale is reduced, thus reach the beneficial effect for reducing industrial production cost,
Production process no longer needs to the chemical substance that addition has risk of causing a disease, and improves the safety of production.
Detailed description of the invention
Fig. 1 is Chinese hamster ovary cell GS gene structure figure.
Fig. 2 is the influence that L-Gln knocks out cell growth to GS.
Fig. 3 is the relational graph that wild-type cell and GS knock out L-Gln in the growth and culture medium of cell.
Fig. 4 is the soda acid distribution map that GS knocks out cell and wild-type cell expresses identical glycoprotein.
Fig. 5 is to knock out cell with GS in 200L scale evaluation as the application of the recombinant protein expression cell of host.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below in conjunction with attached drawing and by specific real
Mode to further illustrate the technical scheme of the present invention is applied, but the present invention is not limited in scope of embodiments.
Embodiment 1: for the building of the CRISPR-Cas9 expression system of GS gene
The GS gene of Chinese hamster ovary cell includes 7 exons, wherein exon 2~7 coding expression GS albumen (figures
1), play the role of to the performance of normal function vital.CrRNA has been separately designed for 2~7 exons of GS gene
Sequence.The GS albumen given expression to is enabled to lose function using these crRNA sequences and Cas9 protein.Exon2~7 of GS
Exon partial sequence is following (exon uses underscore " _ _ _ _ _ " label):
SEQ ID NO.17:
TGCCTCTTACGCAATTCCTGCAGGGGACCCCCTTCAGAGTAGATGTTAATGAAATGACTTTTGTCTCTC
CAGAGCACCTTCCACCATGGCCACCTCAGCAAGTTCCCACTTGAACAAAAACATCAAGCAAATGTACTTGTGCCTGC CCCAGGGTGAGAAAGTCCAAGCCATGTATATCTGGGTTGATGGTACTGGAGAAGGACTGCGCTGCAAAACCCGCACC CTGGACTGTGAGCCCAAGTGTGTAGAAGGTGAGCATGGGCAGGAGCAGGACATGTGCCTGGAAGTGGGCAAGCAGCC
TGAGATTTGACCTTCCTTCTGTTTTGTTTGCAAAGTCTTTCAAAAGCAGGTCTCTTCAGGCCTCAGTCAGTCACCCG
TAAGCTGCCGAGTAGTCTGGAGG
SEQ ID NO.18:
ACTCAGTTACCAGCACCTACATGGTGGCTCACAACCATCTGTAACTCCAATTTCAGGGGCTCCAACCCC
CTCTTCTGCAGGCATACACTTGCACAGATATACATGCAAGTAAAACACCCCTACACACATAAAAATAAATACGTCTT
CTTAAAAGTTAATTTTCCATCTTTATTTGGCCCAGAGTTACCTGAGTGGAATTTTGATGGCTCTAGTACCTTTCAGT CTGAGGGCTCCAACAGTGACATGTATCTCAGCCCTGTTGCCATGTTTCGGGACCCCTTCCGCAGAGATCCCAACAAG CTGGTGTTCTGTGAAGTTTTCAAGTACAACCGGAAGCCTGCAGGTGTGTATGGGGTGGGCGTGAATGTCTTAAGAAT
CTAGGGATGGATGATCAGATGTCCATCCTTCTACCCTGAACTTGCCTGCTGAAAAACAGTGTGGTCCGCCCCTCCAT
GGTCCCTTTTATTGGTTGTATAAACAGTGTTGAATCTTCCATCTGT
SEQ ID NO.19:
TATAAAAAGTCTCTCAGCCTTTTCCTGCAAATGTACTATCATTGCTTCTTCACAGTGGTTGGGCCTGAG
TAGGTCCAGCCTATGATGACTTCAGCTGTGTAAGAGTTGAGGACACTACTCCTTACAGCATGTTGATGCTTTATTCC
TAGAGACCAATTTAAGGCACTCGTGTAAACGGATAATGGACATGGTGAGCAACCAGCACCCCTGGTTTGGAATGGAA CAGGAGTATACTCTGATGGGAACAGATGGGCACCCTTTTGGTTGGCCTTCCAATGGCTTTCCTGGGCCCCAAGGTAA
GTTCCCCAGGTGAAATAAAAGCTTCCTCCCCATAAGTTCTTACTGTCCAGAGACAGGAGCAGCTCCCAAATCAGCAA
ACAGACTGGCAGCTGAAAATAAC
SEQ ID NO.20:
TAGTTCTACCAAGGCTACAGCAAAGAGGAAAGACCTAGCCTCCTACCTGCAGGTGAAGACAGGATGTGC
GAAAGCAAGTCTTAGGACCTTGTCATTTTCTGGCTTTGGGGGGTTATGGACTCTGATTCTTCACTGATTGCTCTTGA
TTCTCCTTCAGGTCCGTATTACTGTGGTGTGGGCGCAGACAAAGCCTATGGCAGGGATATCGTGGAGGCTCACTACC GCGCCTGCTTGTATGCTGGGGTCAAGATTACAGGAACAAATGCTGAGGTCATGCCTGCCCAGGTAAATGGCACTATT
SEQ ID NO.21:
CTGTTCCTTTTCCTCCCCTCTGAAGACTTGGCACATGGGGACTTTGGTTAACAAGGGTGATGACTTAAA
AGTGGTTCAGGGTAGAGGTAAGTAGAACAAGCTAGGAGCTTGAGTTGGCCTGAACAGTTAGTTGGCCTTATTCTAAAGGTCAACATGTTCTTTCTAGTGGGAATTCCAAATAGGACCCTGTGAAGGAATCCGCATGGGAGATCATCTCTGGGTG GCCCGTTTCATCTTGCATCGAGTATGTGAAGACTTTGGGGTAATAGCAACCTTTGACCCCAAGCCCATTCCTGGGAA CTGGAATGGTGCAGGCTGCCATACCAACTTTAGCACCAAGGCCATGCGGGAGGAGAATGGTCTGAAGTAAGTAGCTT
CCTCTGGAGCCATCTTTATTCTCATGGGGTGGAAGGGCTTTGTGTTAGGGTTGGGAAAGTTGGACTTCTCACAAACT
ACATGCCATGCTCTTCGTGTTTGTCATAAGCCTATCGTTTTGTACC
SEQ ID NO.22:
GACTGGTCTGAAGCACTTGAGACATAGGTCACAAGGCAGACACAGCCTGCATCAAGTATTTATTGGTTT
CTTATGGAACTCATGCCTGCTCCTGCCCTTGAAGGACAGGTTTCTAGTGACAAGGTCAGACCCTCACCTTTACTGCT
TCCACCAGGCACATCGAGGAGGCCATCGAGAAACTAAGCAAGCGGCACCGGTACCACATTCGAGCCTACGATCCCAA GGGGGGCCTGGACAATGCCCGTCGTCTGACTGGGTTCCACGAAACGTCCAACATCAACGACTTTTCTGCTGGTGTCG CCAATCGCAGTGCCAGCATCCGCATTCCCCGGACTGTCGGCCAGGAGAAGAAAGGTTACTTTGAAGACCGCCGCCCC TCTGCCAATTGTGACCCCTTTGCAGTGACAGAAGCCATCGTCCGCACATGCCTTCTCAATGAGACTGGCGACGAGCC CTTCCAATACAAAAACTAATTAGACTTTGAGTGATCTTGAGCCTTTCCTAGTTCATCCCACCCCGCCCCAGCTGTCT
CATTGTAACTCAAAGGATGGAATATCAAGGTCTTTTTATTCCTCGTGCCCAGTTAATCTTGCTTTTATTGGTCAGAA
TAGAGGAGTCAAGTTCTTAATCCCTATACACCCAACCCTCATTTCTTTTCTATTTAGCTTTCTAGTGGGGGTGGGAG
GGGTAGGGGAAGGGA
It is following (table 1) for crRNA sequence designed by the above exon:
Table 1
Embodiment 2: the knockout and verifying of GS gene are carried out to CHO-K1 cell
Designed crRNA and Cas9 protein expression vector turn the CHO-K1 cell tamed through CD culture medium
Dye designs A~P totally 16 kinds of transfections or transfection combination altogether.
CHO-K1 cell is purchased from ATCC, and cell culture uses DMEM/F12 basal medium, adds 5% fetal calf serum.
CHO-K1 cell after amplification is cultivated using CD CHO culture medium into suspending, glutamine and HT additive are added.
After cell adapted suspension culture, crRNA and Cas9 protein expression vector are transfected into cell.After transfecting 48hr
Puromycin is added into cell culture medium, after culture 4 days, the cell selection of survival is gone out, subclone culture is carried out.Ya Ke
After grand cell is grown, cell is transferred to the culture medium containing L-Gln and without L-Gln respectively and is cultivated.L- will be free of
It can not be grown in the culture medium of Gln, can be picked out in the culture medium containing L-Gln with the clone of normal growth, carry out amplification training
It supports.
It knocks out cell to A~P GS that totally 16 kinds of transfections or transfection combination are screened to count, statistics indicate that carrying out
3 kinds to 4 kinds target spots knock out jointly, knock out efficiency highest, the results are shown in Table 2.
Table 2
The clone of amplification cultivation is transferred to shaking flask to cultivate.The cell for capableing of normal growth in shaking flask is carried out
Gln removes verifying, filters out under suspended state, grows, it is necessary to rely on the cell (such as Fig. 2-3) of Gln growth.By above
Method, screening obtains 20 plants of GS gene deleted cell strains altogether.
The experiment of embodiment 3:GS gene defect cells expressing antibody
Antibody expression transfection experiment, wild type CHO-K1 cell conduct are carried out to GS KO1 and GS the KO2 cell picked out
Compare host.KJ015 is transfected by identical method to GS KO1, GS KO2, CHO-K1 cell.48hr after transfection,
25 μM of MSX are added as screening pressure.After stationary culture 2 weeks, clone is grown, to the quantity for growing clone and for 24 hours r antibody
Highest 25 clones of expression quantity compare, and the results are shown in Table 3.Clone's quantity variance that three grows is smaller, expression quantity
GS KO1 and GS KO2 are that the obvious expression quantity of clone of host is high.
Table 3
Expression quantity preferably cell is subjected to amplification cultivation, and collects expression product, is analyzed by Key Quality Indicator of IEC
Express the quality of antibody.GS, which knocks out cellular products quality and the quality of wild-type cell product, as the result is shown can reach identical water
Flat (table 4).
Table 4
Embodiment 4:GS gene defect cell expresses glycoprotein experiment
P-glycoprotein expression transfection experiment is carried out to GS KO1 and GS the KO2 cell picked out, wild type CHO-K1 cell is made
To compare host.KJ018 is transfected by identical method to GS KO1, GS KO2, CHO-K1 cell.After transfection
48hr adds 25 μM of MSX as screening pressure.After stationary culture 2 weeks, clone is grown, to the quantity for growing clone and for 24 hours r
P-glycoprotein expression amount is counted, and the results are shown in Table 5.Clone's quantity variance that three grows is smaller, expression quantity GS KO1 and
GS KO2 is that the obvious expression quantity of cell of host is high.
Table 5
Expression quantity preferably cell is subjected to amplification cultivation, and collects expression product, is analyzed by Key Quality Indicator of IEF
Express the quality of glycoprotein.GS, which knocks out cellular products quality and the quality of wild-type cell product, as the result is shown can reach identical
Horizontal (Fig. 4).
Embodiment 5: being the pilot scale culture (200L) of the antibody expressing cells of host with GS gene defect cell
The obtained antibody overexpression cell line GS KO1A5 of screening is subjected to amplification cultivation in shaking flask, when cell is in pair
When number growth period, by cell inoculation to 14L bioreactor, working volume 10L.The cell of reacted device amplification, is seeded to
200L reactor.Cell density is 0.3 × 10 after inoculation6Cells/mL or so, it is 130L, setting that reactor, which originates working volume,
37 DEG C of parameters temperature, pH 6.90-7.20, DO 20%-50%, daily sterile sampling, detects glucose and lactate contains
Amount.The technique used is cultivated as feed supplement batch culture.The index for needing to monitor in incubation has: temperature, pH, concentration of glucose,
Lactate concentration, Morie osmolarity, expression protein concentration.
Cell was at reactor growth 11 days, and antibody expression amount is 5.1g/L, and when terminating culture, cell state is good, vigor
For 98% (Fig. 5).The above result shows that GS deficient cells can be cultivated in 200L scale, and it is able to maintain that good
The high expression and quality of cell state, albumen.According to this results presumption, host's GS deficient cells of the clone are suitable for recombination
The host cell of albumen industrialized production cell.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Claims (12)
1. a kind of for knocking out the CRISPR gene editing system of GS gene, which is characterized in that the gene editing system includes
CrRNA, Cas9 protein expression vector and tracrRNA of special target GS gene；Wherein, the special target GS gene
The sequence of crRNA is the combination of at least two sequences in SEQ ID NO.9-SEQ ID NO.16.
2. a kind of recombinant protein expression host cell, which is characterized in that the host cell line passes through as described in claim 1
System carries out obtained by gene editing the cell.
3. host cell according to claim 2, which is characterized in that the host cell, GS gene lacks functionality.
4. host cell according to claim 2, which is characterized in that the host cell, can high efficient expression include outer
The base sequence of source destination protein.
5. host cell according to claim 4, which is characterized in that the destination protein is recombinant glycoprotein, recombinates and swash
Element, recombination enzyme, any one in recombinant antibodies or antibody fragment.
6. host cell according to claim 5, which is characterized in that the host cell is selected from mammalian cell.
7. host cell according to claim 6, which is characterized in that the mammalian cell is that CHO, NS0 or 293 are thin
Any one in born of the same parents.
8. a kind of method for knocking out GS gene using gene editing system described in claim 1, which is characterized in that including following
Step:
(1) crRNA of selectively targeted GS gene is constructed；
(2) crRNA and tracrRNA and the Cas9 protein expression vector of step (1) are mixed, is transferred in host cell, with life
Produce the cell of GS gene lacks functionality, wherein
The method for being transferred to host cell is electroporation transfection, liposome transfection, any one in nuclear transfection.
9. according to the method described in claim 8, it is characterized in that, after step (2) described mixing, crRNA and tracrRNA's
Molar ratio is 1:1.
10. application of the method as claimed in claim 9 in the cell that preparation knocks out GS gene.
11. application of the host cell according to claim 5 in preparation and reorganization albumen.
12. application according to claim 11, which is characterized in that the recombinant protein is clinical treatment drug.
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| CN110343668B (en) * | 2019-07-23 | 2021-11-19 | 珠海联邦制药股份有限公司 | GS gene knockout CHO cell strain and preparation method and application thereof |
| 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 |
| 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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| CN105392885A (en) * | 2013-07-19 | 2016-03-09 | 赖瑞克斯生物科技公司 | Methods and compositions for producing double allele knock outs |
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Non-Patent Citations (2)
| Title |
|---|
| CRISPR/Cas9-mediated genome engineering of CHO cell factories: Application and perspectives;Jae Seong Lee等;《Biotechnology Journal》;20150731;第10卷(第7期);第979-994页 |
| Improving the Efficiency of CHO Cell Line Generation Using Glutamine Synthetase Gene Knockout Cells;Lianchun Fan等;《Biotechnology and Bioengineering》;20111108;第109卷(第4期);第1007-1015页 |
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