CN109439662A - For the sgRNA of C5aR1 gene knockout, carrier and construction method and detection method - Google Patents
For the sgRNA of C5aR1 gene knockout, carrier and construction method and detection method Download PDFInfo
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Abstract
The present invention relates to gene knockout fields, in particular to for the sgRNA of C5aR1 gene knockout, carrier and construction method and detection method.SgRNA for C5aR1 gene knockout, which is characterized in that the nucleic acid sequence of the sgRNA is as shown in SEQ ID NO.1 and SEQ ID NO.2.The present invention knocks out the analysis of sequence by treating, and relates to 14 sgRNA altogether, by Activity determination, obtains this two sgRNA, verify by subsequent experimental, cooperates with Cas9 albumen, obtains the mouse of C5aR1 gene knockout.
Description
Technical field
The present invention relates to gene knockout field, in particular to for C5aR1 gene knockout sgRNA, carrier with
And construction method and detection method.
Background technique
Chronic obstructive pulmonary disease (Chronic Obstructive Pulmonary Disease, COPD) is a kind of chronic
Airway inflammation disease, Airway Remodeling (airway wall remodeling) is one of its main pathological characteristic and disease
The refractory major reason of disease progression.The chronic inflammation of the generation of chronic obstructive pulmonary disease and air flue and lung to pernicious gas or particle
Disease increased response is related.
It is now recognized that Airway Remodeling is other than abnormal repair process on the basis of airway inflammation, it is also possible to be damaged with oxidation
The mechanism such as the removing exception of wound, protease-antiprotease imbalance and apoptosis and apoptotic cell are related.Although clinically using
Inhaled etc. is directed to the treatment of airway inflammation, but therapeutic effect is still dissatisfied, prompts known COPD Airway Remodeling
Mechanism is still not perfect, needs other mechanism for further clarifying Airway Remodeling, explores and find new therapy target, intervene and
Reverse disease process.
C5a anaphylatoxin (Complement component 5a, C5a) is a member in complement system, a large amount of evidences
Confirm that C5a has played very important effect in acute inflammatory reaction, but its effect in chronic inflammatory reaction rarely has
It refers to.
In view of this, the present invention is specifically proposed.
Summary of the invention
In previous work, inventor has found that air flue induction of sputum C5a level not only rises in the patient of COPD acute exacerbation
Height equally increases in stable COPD patient airway induction of sputum, and C5a is prompted not only to play a role in acute inflammation,
It may also be played an important role in the maintenance and development of the chronic airway inflammation of COPD stationary phase, but its specific effect and work
It is still unclear with mechanism.In depth effect and molecular mechanism of the research C5a in Airway Remodeling, for finding new COPD gas
The therapy target of road remodeling is of great significance.
Based on above content, the purpose of the present invention is to provide the methods of building C5aR1 knock out mice comprising
Designed for the sgRNA and its carrier of C5aR1 gene knockout, and then by injecting corresponding sgRNA and Cas9 albumen, obtain
The mouse of C5aR1 gene knockout, for above-mentioned research.
The present invention provides the sgRNA for C5aR1 gene knockout, the nucleic acid sequence of the sgRNA such as SEQ ID
Shown in NO.1 and SEQ ID NO.2.
The present invention through treating knock out sequence analysis, relate to 14 sgRNA altogether, by Activity determination, obtain this two
SgRNA, is verified by subsequent experimental, is cooperated with Cas9 albumen, is obtained the mouse of C5aR1 gene knockout.
The present invention also provides the T7-sgRNA for C5aR1 gene knockout, the nucleic acid sequence of the T7-sgRNA is such as
Shown in SEQ ID NO.3 and SEQ ID NO.4.
It provided by the present invention for the sgRNA of C5aR1 gene knockout, is transcribed using carrier, is used for the sequence transcribed
Column are transcribed as shown in SEQ ID NO.3 and SEQ ID NO.4, obtain enough sgRNA for injection.
The present invention also provides a kind of carriers, contain above-mentioned T7-sgRNA.
The carrier is used to transcribe the sgRNA for C5aR1 gene knockout, obtains enough sgRNA for injection.
The present invention also provides a kind of methods for constructing C5aR1 knock out mice, as shown in SEQ ID NO.1
SgRNA and Cas9 albumen or the sgRNA as shown in SEQ ID NO.2 and Cas9 albumen are transferred in mouse fertilized egg, subsequent warp
Genotype detection is crossed, the model mice of C5aR1 knockout is obtained.
Further, described to be transferred to by the way of microinjection.
Further, the sgRNA as shown in SEQ ID NO.3 and the sgRNA as shown in SEQ ID NO.4 are connected into respectively
It is transcribed in vitro on expression vector, obtains the sgRNA for microinjection；
The expression vector is the carrier for carrying T7 promoter plasmid.
The present invention also provides the C5aR1 bases that the method for detecting above-mentioned building C5aR1 knock out mice obtains
Because of the primer sequence of knock-out mice, the primer sequence is as shown in SEQ ID NO.5-7.
The primer that the present invention designs, primer sequence such as SEQ ID NO.5-6 cooperation, can only amplify the equipotential of wild type
Gene.
The cooperation of primer sequence such as SEQ ID NO.5 and 7, this is used to confirm the generation of gene knockout, two primers to primer
It separately designs in the two sides for knocking out sequence.Theoretically, for both heterozygote, 2 can be obtained when carrying out PCR to primer using this
A product: PCR product, the PCR product of mutant allele of wild-type allele；But in fact, the two products
Difference is very big sometimes for length, and the PCR product of wild-type allele is long, and the PCR product of mutant allele compares
It is short, the PCR product on wild-type allele possibly can not be amplified when PCR, cannot also confirm the specific genotype of animal
(including wild type, heterozygosis, homozygosis), and not can confirm that specific location and mutation alkali of the mutant gene sequence in genome
Radix.
In conjunction with above-mentioned primer as a result, the specific genotype of animal: homozygote/heterozygote/wild can be determined effectively
Type.
The present invention also provides a kind of C5aR1 bases that the method for detecting above-mentioned building C5aR1 knock out mice obtains
Because of the kit of knock-out mice, contain above-mentioned primer sequence.
Further, the kit further includes any one of dNTPs, archaeal dna polymerase, buffer, distilled water or several
Kind.
Kit provided by the invention, the detection for C5aR1 knock out mice provide convenience.
The present invention also provides a kind of C5aR1 bases that the method for detecting above-mentioned building C5aR1 knock out mice obtains
Because of the method for knock-out mice, above-mentioned primer sequence is carried out PCR amplification, is obtained using the genome of object to be detected as template
Product sequencing, judges the genotype of the detection site of the object to be detected.
Further, the object to be detected is rat-tail genome.
Further, PCR amplification system is 20-25 μ L.
Further, the annealing temperature of PCR amplification is 62 DEG C.
Compared with prior art, the invention has the benefit that
(1) present invention knocks out the analysis of sequence by treating, and relates to 14 sgRNA altogether and obtains by Activity determination
This two sgRNA, are verified by subsequent experimental, are cooperated with Cas9 albumen, are obtained the mouse of C5aR1 gene knockout.
(2) the present invention also provides the T7-sgRNA for C5aR1 gene knockout, are connected into carrier and are transcribed, are obtained
Enough sgRNA for injection.
(3) the present invention also provides a kind of method for constructing C5aR1 knock out mice, method is easy, can effectively obtain
The mouse of C5aR1 gene knockout.
(4) the present invention also provides the primer, kit and method for detecting C5aR1 knock out mice, it is
The detection of C5aR1 knock out mice provides convenience.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below will to embodiment or
Attached drawing needed to be used in the description of the prior art is briefly described.
Fig. 1 is EGE-WFZ-004 gene knockout strategy schematic diagram in the embodiment of the present invention 1；
Fig. 2 is the Activity determination result figure of Cas9/sgRNA in the embodiment of the present invention 1；
Fig. 3 is the electrophoresis detection figure that sgRNA is transcribed in vitro in the embodiment of the present invention 2 on T7 promoter plasmid carrier；
Fig. 4 is that design of primers schematic diagram is identified in the embodiment of the present invention 2；
Fig. 5 is F0 in the embodiment of the present invention 2 for Genotyping testing result figure；
Fig. 6 is F1 generation Genotyping EGE-WFZ-004-WT-F/EGE-WFZ-004-Mut-R inspection in the embodiment of the present invention 2
Survey result figure；
Fig. 7 is F1 generation Genotyping EGE-WFZ-004-WT-F/EGE-WFZ-004-WT-R detection knot in the embodiment of the present invention 2
Fruit figure.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
It will be appreciated that the following example is merely to illustrate the present invention, and it is not construed as limiting the scope of the invention.It is not specified in embodiment
Actual conditions person carries out according to conventional conditions or manufacturer's recommended conditions.Production firm is not specified in agents useful for same or instrument
Person is the conventional products that can be obtained by commercially available purchase.
Embodiment 1
The design of EGE-WFZ-004 gene knockout mode mouse
1, the understanding of relevant information and strategy design of practicing shooting
EGE-WFZ-004 gene is on No. 7 chromosome anti-chains, overall length about 12.59kb.Gene ID: 12273.EGE-
WFZ-004 gene has 3 transcripts.
EGE-WFZ-004 gene structure is analyzed, entire code area is deleted in selection.SgRNAs is separately designed
The non-conservative area in the downstream the UTR of Intron1-2 and 3 ', about causes the genomic deletion of 5.5kb, to reach EGE-WFZ-004
The purpose of gene knockout.The EGE system preparation mode mouse developed using hundred Olympic Competition figure companies based on CRISPR/Cas9.Specifically
Knock out strategy as shown in Figure 1.
2, the preparation of EGE-WFZ-004 gene knockout mode mouse
The sequencing of 2.1 target sequences confirms
Different lines, target-gene sequence may differences.In order to guarantee the efficiency of designed Cas9/sgRNA, first
It needs to carry out PCR amplification and sequence verification to C57BL/6 rat-tail target site sequence, to guarantee sgRNA identification sequence and C57BL/
6 rat-tail DNA sequence dnas are completely the same.PCR primer is as shown in table 1.
1 PCR primer of table
PCR and sequencing are carried out to C57BL/6 rat-tail DNA, as a result proved: C57BL/6 rat-tail target sequence and Genebank and
Ensembl is given, and sequence is completely the same.
2.2 Cas9/sgRNA design and Activity determination
2.2. the design of 1Cas9/sgRNA
Design principle based on sgRNA respectively designs 7 sgRNA in 5 ' target sites and 3 ' target site regions, such as table 2 and table
Shown in 3.
25 ' target site of table
33 ' target site of table
The adjustment of sequence is carried out to the sgRNA that table 2 and table 3 design, obtained sequence is as shown in table 4.
The sequence of 4 sgRNA of table
| sgRNA | GuideRNA sequence |
| EGE-WFZ-004-sgRNA1 | GGATCCTGGTGTCCTCGGT |
| EGE-WFZ-004-sgRNA2 | GGTCACATTTCCAGCCCACCG |
| EGE-WFZ-004-sgRNA3 | GGCCCAAAGTTGGTATAAAGAC |
| EGE-WFZ-004-sgRNA4 | GGAACTGTGACCGATATCTG |
| EGE-WFZ-004-sgRNA5 | GGCCCAATATGTTTATACACCA |
| EGE-WFZ-004-sgRNA6 | GGTCTTTATACCAACTTT |
| EGE-WFZ-004-sgRNA7 | GGAGTAGCAAGCTTACAGTC |
| EGE-WFZ-004-sgRNA8 | GGATTGATGGTAGGGTGCTTA |
| EGE-WFZ-004-sgRNA9 | GGCCAGGTGTCCCCTCCTTAA |
| EGE-WFZ-004-sgRNA10 | GGCACAGGGGACTCCCTTAAGG |
| EGE-WFZ-004-sgRNA11 | GGCTGTAAAACCAGCTTA |
| EGE-WFZ-004-sgRNA12 | GGCCACAAGAGGGAGACAACT |
| EGE-WFZ-004-sgRNA13 | GGCAGCCATTACAAATCATT |
| EGE-WFZ-004-sgRNA14 | GGCATGTATGTCCAGCATGTG |
2.2.2 the building of Cas9/sgRNA plasmid
The corresponding primer of sgRNA sequent synthesis is designed according to table 4, is connected into pCS-3G by way of Gibson Assembly
Carrier, sample presentation sequence verification is correct after connection product conversion.
The Activity determination of 2.3 Cas9/sgRNA
The Activity determination of sgRNA is carried out using the CRISPR/Cas9 activity test method-UCATM mode of hundred Olympic Competition figures.
(specific experiment method is detailed in company to the advantages that it is limited with no species, high-throughput, wide adaptability, high sensitivity, simplicity
Website).
Obtained testing result is as shown in Figure 2.
Comprehensively consider, EGE-WFZ-004-sgRNA4 and EGE-WFZ-004-sgRNA13 is selected to carry out next step experiment.
Embodiment 2
EGE-WFZ-004-sgRNA4 and the EGE-WFZ-004-sgRNA13 design that embodiment 1 obtains are connected into band T7 and start
The sequence being transcribed in vitro on sub- plasmid vector, specific as follows:
EGE-WFZ-004-T7-sgRNA4:
CTATTTCTAGCTCTAAAACCAGATATCGGTCACAGTTCCTATAGTGAGT CGTATTA；
EGE-WFZ-004-T7-sgRNA13:
CTATTTCTAGCTCTAAAACAATGATTTGTAATGGCTGCCTATAGTGAGT CGTATTA。
EGE-WFZ-004-T7-sgRNA4 and EGE-WFZ-004-T7-sgRNA13 is connected into band T7 promoter plasmid respectively
It is transcribed on carrier, obtained RNA is as shown in Figure 3.Obtained RNA is used for microinjection.
1, the microinjection of Cas9/sgRNA
By Cas9/sgRNA difference microinjection into mouse (C57BL/6N) fertilized eggs, co-injection 332, after injection
The birth of F0 mouse has 45.
2, F0 is detected for murine genes type
Identify that design of primers is as shown in Figure 4.In Fig. 4, arrow represents the position and direction of design of primers. EGE-WFZ-
004-WT-F/EGE-WFZ-004-Mut-R: this is used to confirm the generation of gene knockout to primer, and two primers separately design
Knock out the two sides of sequence.Theoretically, for both heterozygote, 2 products can be obtained when carrying out PCR to primer using this: wild
PCR product, the PCR product of mutant allele of type allele；But in fact, the length of the two products has the time difference
Very not big, the PCR product of wild-type allele is long, and the PCR product of mutant allele is shorter, when PCR
PCR product on wild-type allele possibly can not be amplified, cannot also confirm specific genotype (including the open country of animal
Raw type, heterozygosis, homozygosis), and not can confirm that specific location and mutating alkali yl number of the mutant gene sequence in genome.
EGE-WFZ-004-WT-F/EGE-WFZ-004-WT-R: this can only amplify wild-type allele to primer
PCR product, in conjunction with EGE-WFZ-004-WT-F/EGE-WFZ-004-Mut-R PCR as a result, the specific of animal will can be determined
Genotype: homozygote/heterozygote/wild type.
Obtained primer is as shown in table 5.
5 primer sequence of table
Note: WT- wild-type allele；Mut- mutant allele.
PCR condition:
Enzyme:KOD-FX, system are 20 μ l.
Primer: EGE-WFZ-004-WT-F/EGE-WFZ-004-Mut-R, obtained PCR product detection figure such as Fig. 5 institute
Show.
By PCR experiment and sequencing result, show #E3Z4-003, #E3Z4-004, #E3Z4-006, #E3Z4-008, #
E3Z4-009,#E3Z4-012,#E3Z4-017,#E3Z4-018,#E3Z4-022, #E3Z4-026,#E3Z4-028,#E3Z4-
031,#E3Z4-032,#E3Z4-033,#E3Z4-035, #E3Z4-036,#E3Z4-037,#E3Z4-040and#E3Z4-045
It is F0 for positive mice.
Quickly due to embryo's Early cleavage speed, obtained F0 mouse is chimera.Therefore with the progress of F0 mouse rat-tail
It identifies that obtained F0 genotype is only for reference, one cannot be represented and be set to heritable genic mutation type, heritable genotype
It need to be determined after the detection of F1 mouse rat-tail.
3, F1 generation murine genes type is identified
Above-mentioned positive F0 mouse is mated respectively to obtain with wild-type mice has the F1 for stablizing genotype for mouse, hands over
With the results are shown in Table 6.
The mating result of table 6
Note: "-" expression does not count.
Detection primer used is same as above, unlike, PCR condition:
Enzyme:Taq。
Testing result is as shown in Figures 6 and 7.In Fig. 6, Primers:EGE-WFZ-004-WT-F/EGE-WFZ-004-Mut-
R；In Fig. 7, Primers:EGE-WFZ-004-WT-F/EGE-WFZ-004-WT-R.
Through being sequenced, the F1 generation positive gene parting in Fig. 6 is as shown in table 7.
7 F1 generation positive gene parting of table
| Mouse ID | Father ID | Mother ID | Gender | Genotype |
| 1E3Z4-006 | E3Z4-003 | C57BL/6N | ♀ | △5468in3/+ |
| 1E3Z4-008 | E3Z4-003 | C57BL/6N | ♀ | △5468in3/+ |
| 1E3Z4-011 | E3Z4-033 | C57BL/6N | ♂ | △5469in6/+ |
| 1E3Z4-016 | E3Z4-033 | C57BL/6N | ♀ | △5469in6/+ |
| 1E3Z4-018 | E3Z4-033 | C57BL/6N | ♀ | △5469in6/+ |
| 1E3Z4-020 | E3Z4-033 | C57BL/6N | ♀ | △5469in6/+ |
By PCR identification and sequencing result, show 1E3Z4-006,1E3Z4-008,1E3Z4-011,1E3Z4-016,
1E3Z4-018,1E3Z4-020 is F1 generation positive mice.Wherein, 1E3Z4-006 and 1E3Z4-008 has lacked 5468 bases,
3 bases are additionally inserted；1E3Z4-011,1E3Z4-016,1E3Z4-018,1E3Z4-020 have lacked 5469 bases, volume
6 bases are inserted outside.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>The Third Affiliated Hospital of Peking University
<120>sgRNA, carrier and the construction method and detection method of C5aR1 gene knockout are used for
<130> 2018
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
ggaactgtga ccgatatctg 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
ggcagccatt acaaatcatt 20
<210> 3
<211> 56
<212> DNA
<213>artificial sequence
<400> 3
ctatttctag ctctaaaacc agatatcggt cacagttcct atagtgagtc gtatta 56
<210> 4
<211> 56
<212> DNA
<213>artificial sequence
<400> 4
ctatttctag ctctaaaaca atgatttgta atggctgcct atagtgagtc gtatta 56
<210> 5
<211> 25
<212> DNA
<213>artificial sequence
<400> 5
ctgggtttgg agtctgtggc ttcat 25
<210> 6
<211> 25
<212> DNA
<213>artificial sequence
<400> 6
tccactgtat accaggctag tccca 25
<210> 7
<211> 24
<212> DNA
<213>artificial sequence
<400> 7
ctgaaccaga ggcttgtgcg tgtt 24
Claims (10)
1. be used for C5aR1 gene knockout sgRNA, which is characterized in that the nucleic acid sequence of the sgRNA such as SEQ ID NO.1 and
Shown in SEQ ID NO.2.
2. being used for the T7-sgRNA of C5aR1 gene knockout, which is characterized in that the nucleic acid sequence of the T7-sgRNA such as SEQ ID
Shown in NO.3 and SEQ ID NO.4.
3. a kind of carrier, which is characterized in that contain T7-sgRNA as claimed in claim 2.
4. it is a kind of construct C5aR1 knock out mice method, which is characterized in that the sgRNA as shown in SEQ ID NO.1 with
Cas9 albumen or the sgRNA as shown in SEQ ID NO.2 and Cas9 albumen are transferred in mouse fertilized egg, subsequent to pass through genotype
Detection obtains the model mice of C5aR1 knockout.
5. according to the method described in claim 4, it is characterized in that, described be transferred to by the way of microinjection.
6. according to the method described in claim 4, it is characterized in that, the sgRNA as shown in SEQ ID NO.3 and such as SEQ ID
SgRNA shown in NO.4 is connected on expression vector respectively and is transcribed in vitro, and obtains the sgRNA for microinjection；
The expression vector is the carrier for carrying T7 promoter plasmid.
7. the C5aR1 base that the method for detecting the described in any item building C5aR1 knock out mice of claim 4-6 obtains
Because of the primer sequence of knock-out mice, which is characterized in that the primer sequence is as shown in SEQ ID NO.5-7.
8. a kind of C5aR1 base that the method for the described in any item building C5aR1 knock out mice of detection claim 4-6 obtains
Because of the kit of knock-out mice, which is characterized in that contain primer sequence as claimed in claim 7.
9. kit according to claim 8, which is characterized in that the kit further includes dNTPs, archaeal dna polymerase, delays
Any one or more of fliud flushing, distilled water.
10. a kind of C5aR1 that the method for the described in any item building C5aR1 knock out mice of detection claim 4-6 obtains
The method of knock out mice, which is characterized in that the primer sequence in claim 7 is using the genome of object to be detected as mould
Plate carries out PCR amplification, and obtained product sequencing judges the genotype of the detection site of the object to be detected；
Further, the object to be detected is rat-tail genome；
Further, PCR amplification system is 20-25 μ L；
Further, the annealing temperature of PCR amplification is 62 DEG C.
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