CN109439662B - sgRNA for C5aR1 gene knockout, vector, construction method and detection method - Google Patents
sgRNA for C5aR1 gene knockout, vector, construction method and detection method Download PDFInfo
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Abstract
The invention relates to the field of gene knockout, in particular to sgRNA and a vector for knocking out a C5aR1 gene, a construction method and a detection method. sgRNA used for knocking out C5aR1 gene, and is characterized in that the nucleic acid sequence of the sgRNA is shown as SEQ ID NO.1 and SEQ ID NO. 2. According to the invention, 14 sgRNAs are involved in total through analysis of sequences to be knocked out, the two sgRNAs are obtained through activity detection, and the two sgRNAs are matched with Cas9 protein through subsequent experimental verification to obtain a C5aR1 gene knocked-out mouse.
Description
Technical Field
The invention relates to the field of gene knockout, in particular to sgRNA and a vector for knocking out a C5aR1 gene, a construction method and a detection method.
Background
Chronic Obstructive Pulmonary Disease (COPD) is a Chronic airway inflammatory Disease, and airway remodeling (airway wall remodelling) is one of the main pathological features of COPD and an important reason for the difficulty in curing the Disease. Chronic obstructive pulmonary disease occurs in association with an increased chronic inflammatory response of the airways and lungs to harmful gases or particles.
It is currently believed that airway remodeling may be associated with mechanisms such as oxidative damage, protease-anti-protease imbalance, and abnormal clearance of apoptotic and apoptotic cells, in addition to abnormal repair processes based on airway inflammation. Although clinically used for treating airway inflammation by inhaled glucocorticoids and the like, the treatment effect is still unsatisfactory, which suggests that the known mechanism of airway remodeling of COPD is still incomplete, and further definition of other mechanisms of airway remodeling is urgently needed, and a new treatment target is explored and searched, and the disease process is intervened and reversed.
The anaphylatoxin C5a (comparative component 5a, C5a) is a member of the Complement system, and a large body of evidence demonstrates that C5a plays a very important role in acute inflammatory reactions, but its role in chronic inflammatory reactions is rarely mentioned.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
In earlier work, the inventors found that airway-induced sputum C5a levels were elevated not only in patients with acute exacerbation of COPD, but also in patients with COPD in the stationary phase, suggesting that C5a plays a role not only in acute inflammation, but may also have an important role in the maintenance and development of chronic airway inflammation in the stationary phase of COPD, but the specific roles and mechanisms of action thereof are still unclear. The deep research on the role and molecular mechanism of C5a in airway remodeling is of great significance for searching a novel therapeutic target point for COPD airway remodeling.
Based on the above, the invention aims to provide a method for constructing a C5aR1 knockout mouse, which comprises designing sgrnas and vectors thereof for C5aR1 knockout, and further obtaining a C5aR1 knockout mouse by injecting corresponding sgrnas and Cas9 proteins for the above-mentioned research.
The invention provides sgRNA for knocking out a C5aR1 gene, wherein the nucleic acid sequence of the sgRNA is shown in SEQ ID No.1 and SEQ ID No. 2.
According to the invention, 14 sgRNAs are involved in total through analysis of sequences to be knocked out, the two sgRNAs are obtained through activity detection, and the two sgRNAs are matched with Cas9 protein through subsequent experimental verification to obtain a C5aR1 gene knocked-out mouse.
The invention also provides T7-sgRNA for knocking out the C5aR1 gene, wherein the nucleic acid sequence of the T7-sgRNA is shown as SEQ ID NO.3 and SEQ ID NO. 4.
The sgRNA for knocking out the C5aR1 gene provided by the invention is transcribed by adopting a vector, the sequence for transcription is shown as SEQ ID No.3 and SEQ ID No.4, and sufficient sgRNA for injection is obtained through transcription.
The invention also provides a vector containing the T7-sgRNA.
The vector is used for transcribing sgrnas for C5aR1 gene knockout, and sufficient amount of sgrnas for injection are obtained.
The invention also provides a method for constructing a C5aR1 knockout mouse, wherein sgRNA and Cas9 protein shown in SEQ ID No.1 or sgRNA and Cas9 protein shown in SEQ ID No.2 are transferred into mouse fertilized eggs, and a C5aR1 knockout model mouse is obtained through genotype detection.
Further, the transfer was carried out by microinjection.
Further, the sgRNA shown in SEQ ID No.3 and the sgRNA shown in SEQ ID No.4 are respectively connected to an expression vector for in vitro transcription to obtain the sgRNA for microinjection;
the expression vector is a vector carrying a T7 promoter plasmid.
The invention also provides a primer sequence of the C5aR1 gene knockout mouse obtained by the method for constructing the C5aR1 gene knockout mouse, wherein the primer sequence is shown as SEQ ID NO. 5-7.
The primer sequence of the primer designed by the invention is matched as SEQ ID NO.5-6, and only the wild allele can be amplified.
The primer sequences are matched as SEQ ID NO.5 and 7, the pair of primers is used for confirming the occurrence of gene knockout, and the two primers are respectively designed on two sides of the knockout sequence. Theoretically, for heterozygote animals, PCR using this pair of primers would yield 2 products: PCR products of wild type alleles, PCR products of mutant alleles; however, in practice, the lengths of these two products are sometimes very different, the PCR product of the wild-type allele is relatively long, and the PCR product of the mutant allele is relatively short, so that the PCR product of the wild-type allele may not be amplified during PCR, and thus the specific genotype (including wild-type, heterozygous, homozygous) of the animal may not be confirmed, and the specific position and the number of mutation bases of the mutant gene sequence in the genome may not be confirmed.
The result of combining the primers can effectively determine the specific genotype of the animal: homozygote/heterozygote/wild type.
The invention also provides a kit for detecting the C5aR1 gene knockout mouse obtained by the method for constructing the C5aR1 gene knockout mouse, and the kit contains the primer sequence.
Further, the kit also comprises one or more of dNTPs, DNA polymerase, buffer solution and double distilled water.
The kit provided by the invention provides convenience for detecting the C5aR1 gene knockout mouse.
The invention also provides a method for detecting the C5aR1 gene knockout mouse obtained by the method for constructing the C5aR1 gene knockout mouse, wherein the primer sequence takes the genome of an object to be detected as a template, PCR amplification is carried out, the obtained product is sequenced, and the genotype of the detection site of the object to be detected is judged.
Further, the object to be detected is a rat tail genome.
Further, the PCR amplification system is 20-25. mu.L.
Further, the annealing temperature for PCR amplification was 62 ℃.
Compared with the prior art, the invention has the beneficial effects that:
(1) according to the invention, 14 sgRNAs are involved in total through analysis of sequences to be knocked out, the two sgRNAs are obtained through activity detection, and the two sgRNAs are matched with Cas9 protein through subsequent experimental verification to obtain a C5aR1 gene knocked-out mouse.
(2) The invention also provides T7-sgRNA for knocking out the C5aR1 gene, which is connected into a vector for transcription to obtain enough sgRNA for injection.
(3) The invention also provides a method for constructing the C5aR1 gene knockout mouse, which is simple and convenient and can effectively obtain the C5aR1 gene knockout mouse.
(4) The invention also provides a primer, a kit and a method for detecting the C5aR1 gene knockout mouse, and provides convenience for detecting the C5aR1 gene knockout mouse.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a schematic diagram showing the EGE-WFZ-004 gene knockout strategy in example 1 of the present invention;
FIG. 2 is a graph showing the results of activity detection of Cas9/sgRNA in example 1 of the present invention;
fig. 3 is an electrophoretic detection map of sgRNA transcribed in vitro on a T7 promoter plasmid vector in example 2 of the present invention;
FIG. 4 is a schematic diagram of the design of the identifying primer in example 2 of the present invention;
FIG. 5 is a graph showing the results of genotyping assay for generation F0 in example 2 of the present invention;
FIG. 6 is a diagram showing the result of detection of EGE-WFZ-004-WT-F/EGE-WFZ-004-Mut-R for genotyping of F1 in example 2 of the present invention;
FIG. 7 is a diagram showing the results of detection of EGE-WFZ-004-WT-F/EGE-WFZ-004-WT-R for genotyping the F1 generation in example 2 of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
Design of EGE-WFZ-004 gene knockout model mouse
1. Knowledge of relevant information and targeting strategy design
The EGE-WFZ-004 gene is located on the reverse strand of chromosome 7 and has a total length of about 12.59 kb. Gene ID: 12273. There are 3 transcripts of the EGE-WFZ-004 gene.
The EGE-WFZ-004 gene structure was analyzed and the entire coding region was selected for deletion. sgRNAs are respectively designed in non-conserved regions at the downstream of Intron1-2 and 3' UTR, and genome deletion of 5.5kb is caused, so that the purpose of EGE-WFZ-004 gene knockout is achieved. Model mice were prepared using the EGE system developed by the paoexel company based on CRISPR/Cas 9. A specific knockout strategy is shown in figure 1.
2. Preparation of EGE-WFZ-004 gene knockout model mouse
2.1 sequencing confirmation of target sequences
The target gene sequence may differ from strain to strain. In order to ensure the efficiency of the designed Cas9/sgRNA, the C57BL/6 rat tail target site sequence needs to be PCR amplified and sequence verified to ensure that the sgRNA recognition sequence is completely consistent with the C57BL/6 rat tail DNA sequence. The PCR primers are shown in Table 1.
TABLE 1 PCR primers
PCR and sequencing were performed on C57BL/6 rat tail DNA, and the results demonstrated that: the C57BL/6 rat tail target sequence was identical to the sequences given by Genebank and Ensembl.
2.2 Cas9/sgRNA design and Activity detection
2.2.1 design of Cas9/sgRNA
Based on the design principle of sgrnas, 7 sgrnas were designed in each of the 5 'target site and 3' target site regions, as shown in tables 2 and 3.
TABLE 25' target sites
TABLE 33' target sites
The sequences of sgrnas designed in tables 2 and 3 were adjusted to obtain sequences shown in table 4.
Table 4 sequences of sgrnas
| 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 construction of Cas9/sgRNA plasmid
The sgRNA sequences are designed according to the table 4, corresponding primers are synthesized, the sgRNA sequences are connected into a pCS-3G vector in a Gibson Assembly mode, and the connection products are transmitted to a sample after being transformed and sequenced to verify the correctness.
2.3 detection of Cas9/sgRNA Activity
The activity of sgRNA is detected by adopting a CRISPR/Cas9 activity detection method of a hundred-Oersatz chart-UCATM mode. The method has the advantages of no species limitation, high flux, wide adaptability, high sensitivity, simplicity and the like (the specific experimental method is detailed in company websites).
The obtained detection results are shown in fig. 2.
In general, EGE-WFZ-004-sgRNA4 and EGE-WFZ-004-sgRNA13 were selected for further experiments.
Example 2
EGE-WFZ-004-sgRNA4 and EGE-WFZ-004-sgRNA13 obtained in example 1 were used to design sequences for in vitro transcription linked to a plasmid vector carrying the T7 promoter, as follows:
EGE-WFZ-004-T7-sgRNA4：
CTATTTCTAGCTCTAAAACCAGATATCGGTCACAGTTCCTATAGTGAGTCGTATTA；
EGE-WFZ-004-T7-sgRNA13：
CTATTTCTAGCTCTAAAACAATGATTTGTAATGGCTGCCTATAGTGAGTCGTATTA。
EGE-WFZ-004-T7-sgRNA4 and EGE-WFZ-004-T7-sgRNA13 are respectively linked to a plasmid vector with a T7 promoter for transcription, and the obtained RNA is shown in figure 3. The resulting RNA was used for microinjection.
1. Microinjection of Cas9/sgRNA
Cas9/sgRNA were microinjected into mouse (C57BL/6N) zygotes for a total of 332, 45F 0 mice were born after injection.
2. Genotype detection of F0 mouse
The identifying primer design is shown in FIG. 4. In FIG. 4, the arrows indicate the position and direction of primer design. EGE-WFZ-004-WT-F/EGE-WFZ-004-Mut-R: the pair of primers is used for confirming the occurrence of gene knockout, and the two primers are respectively designed on two sides of a knockout sequence. Theoretically, for heterozygote animals, PCR using this pair of primers would yield 2 products: PCR products of wild type alleles, PCR products of mutant alleles; however, in practice, the lengths of these two products are sometimes very different, the PCR product of the wild-type allele is relatively long, and the PCR product of the mutant allele is relatively short, so that the PCR product of the wild-type allele may not be amplified during PCR, and thus the specific genotype (including wild-type, heterozygous, homozygous) of the animal may not be confirmed, and the specific position and the number of mutation bases of the mutant gene sequence in the genome may not be confirmed.
EGE-WFZ-004-WT-F/EGE-WFZ-004-WT-R: the pair of primers can only amplify PCR products of wild alleles, and the specific genotype of the animal can be determined by combining the PCR result of EGE-WFZ-004-WT-F/EGE-WFZ-004-Mut-R: homozygote/heterozygote/wild type.
The obtained primers are shown in Table 5.
TABLE 5 primer sequences
Note: WT-wild type allele; mut-mutant allele.
PCR conditions were as follows:
enzyme: KOD-FX in 20. mu.l.
Primer: the detection chart of the PCR product obtained by EGE-WFZ-004-WT-F/EGE-WFZ-004-Mut-R is shown in FIG. 5.
As a result of PCR experiments and sequencing, it was revealed that # 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 were F0 generation positive mice.
The F0 mouse was obtained as a chimera due to the rapid rate of early cleavage of the embryo. Therefore, the F0 genotype identified from the mouse tail of the F0 mouse is only used as a reference and cannot represent a certain heritable gene mutant type, and the heritable genotype needs to be determined after the mouse tail of the F1 mouse is detected.
3. Genotype identification of F1 mouse
The positive F0 mice were mated with wild-type mice to obtain F1 generation mice with stable genotypes, and the mating results are shown in Table 6.
TABLE 6 mating results
Note: "-" indicates not statistically.
The primers used for the detection were as above, except that the PCR conditions:
Enzyme:Taq。
the results of the tests are shown in FIGS. 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.
Sequencing, positive genotyping at F1 in FIG. 6 is shown in Table 7.
TABLE 7F 1 Positive genotyping
The results of PCR identification and sequencing show that 1E3Z4-006,1E3Z4-008,1E3Z4-011,1E3Z4-016,1E3Z4-018 and 1E3Z4-020 are positive F1 mice. Wherein, 5468 bases are deleted and 3 bases are additionally inserted in 1E3Z4-006 and 1E3Z 4-008; 1E3Z4-011,1E3Z4-016,1E3Z4-018 and 1E3Z4-020 lack 5469 bases and are additionally inserted with 6 bases.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
SEQUENCE LISTING
<110> third Hospital of Beijing university
<120> sgRNA for C5aR1 gene knockout, vector, construction method and detection method
<130>2018
<160>7
<170>PatentIn version 3.3
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<213> Artificial sequence
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<210>4
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<213> Artificial sequence
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ctatttctag ctctaaaaca atgatttgta atggctgcct atagtgagtc gtatta 56
<210>5
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<213> Artificial sequence
<400>5
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Claims (6)
1. sgRNA used for knocking out C5aR1 gene, and is characterized in that the nucleic acid sequence of the sgRNA is shown as SEQ ID NO.1 and SEQ ID NO. 2.
2. A method for constructing a C5aR1 gene knockout mouse is characterized in that sgRNA shown in SEQ ID No.1 and sgRNA shown in SEQ ID No.2 are respectively connected to an expression vector for in vitro transcription to obtain sgRNA for microinjection;
the expression vector is a vector carrying a T7 promoter plasmid.
3. The primer sequence of the C5aR1 knockout mouse obtained by the method for constructing the C5aR1 knockout mouse according to the claim 2 is shown as SEQ ID NO. 5-7.
4. A kit for detecting the C5aR1 knockout mouse obtained by the method for constructing the C5aR1 knockout mouse as claimed in claim 2, wherein the kit comprises the primer sequence as claimed in claim 3.
5. The kit according to claim 4, wherein the kit further comprises any one or more of dNTPs, DNA polymerase, buffer solution and double distilled water.
6. A method for detecting the C5aR1 gene knockout mouse obtained by the method for constructing the C5aR1 gene knockout mouse in the claim 3 is characterized in that a primer sequence in the claim 3 takes a genome of an object to be detected as a template, PCR amplification is carried out, an obtained product is sequenced, and the genotype of a detection site of the object to be detected is judged; the object to be detected is a rat tail genome; the PCR amplification system is 20-25 mu L; the annealing temperature for PCR amplification was 62 ℃.
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