CN109439662B - sgRNA for C5aR1 gene knockout, vector, construction method and detection method - Google Patents

Abstract

The present invention relates to the field of gene knockout, in particular, to sgRNA, vector, construction method and detection method for C5aR1 gene knockout. The sgRNA used for C5aR1 gene knockout is characterized in that the nucleic acid sequence of the sgRNA is shown in SEQ ID NO.1 and SEQ ID NO.2. After the analysis of the sequence to be knocked out in the present invention, a total of 14 sgRNAs are involved. After activity detection, these two sgRNAs are obtained. After subsequent experimental verification, they cooperate with the Cas9 protein to obtain a C5aR1 gene knockout mouse.

Description

sgRNA, vector, construction method and detection method for C5aR1 gene knockout

technical field

The present invention relates to the field of gene knockout, in particular, to sgRNA, vector, construction method and detection method for C5aR1 gene knockout.

Background technique

Chronic Obstructive Pulmonary Disease (COPD) is a chronic airway inflammatory disease. Airway wall remodeling is one of its main pathological features and an important reason for disease progression and refractory disease. The development of COPD is associated with an enhanced chronic inflammatory response of the airways and lungs to noxious gases or particles.

At present, it is believed that in addition to the abnormal repair process based on airway inflammation, airway remodeling may also be related to oxidative damage, protease-antiprotease imbalance, and abnormal clearance of apoptotic and apoptotic cells. Although inhaled glucocorticoids and other treatments for airway inflammation are clinically used, the therapeutic effect is still unsatisfactory, suggesting that the known COPD airway remodeling mechanism is still incomplete. It is urgent to further clarify other mechanisms of airway remodeling and explore and finding new therapeutic targets to intervene and reverse disease processes.

Anaphylatoxin C5a (Complement component 5a, C5a) is a member of the complement system. A large number of evidences confirm that C5a plays a very important role in acute inflammatory response, but its role in chronic inflammatory response is rarely mentioned.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

In the previous work, the inventors found that the level of C5a in airway-induced sputum not only increased in patients with acute exacerbation of COPD, but also increased in airway-induced sputum in patients with stable COPD, suggesting that C5a not only plays a role in acute inflammation, but also in It may also play an important role in the maintenance and development of chronic airway inflammation in stable COPD, but its specific role and mechanism remain unclear. In-depth study of the role and molecular mechanism of C5a in airway remodeling is of great significance for finding new therapeutic targets for COPD airway remodeling.

Based on the above content, the purpose of the present invention is to provide a method for constructing a C5aR1 knockout mouse, which includes designing an sgRNA for C5aR1 knockout and a carrier thereof, and then injecting the corresponding sgRNA and Cas9 protein to obtain a C5aR1 knockout mice were used for the above studies.

The present invention provides sgRNA for C5aR1 gene knockout, and the nucleic acid sequence of the sgRNA is shown in SEQ ID NO.1 and SEQ ID NO.2.

After the analysis of the sequence to be knocked out, the present invention involves a total of 14 sgRNAs. After activity detection, these two sgRNAs are obtained. After subsequent experimental verification, they cooperate with the Cas9 protein to obtain a C5aR1 gene knockout mouse.

The present invention also provides T7-sgRNA for C5aR1 gene knockout, and the nucleic acid sequences of the T7-sgRNA are shown in SEQ ID NO.3 and SEQ ID NO.4.

The sgRNA for C5aR1 gene knockout provided by the present invention is transcribed using a vector, and the sequences used for transcription are shown in SEQ ID NO.3 and SEQ ID NO.4. After transcription, a sufficient amount of sgRNA for injection is obtained .

The present invention also provides a vector containing the above-mentioned T7-sgRNA.

This vector is used to transcribe sgRNA for C5aR1 gene knockout, resulting in sufficient sgRNA for injection.

The present invention also provides a method for constructing a C5aR1 knockout mouse, wherein the sgRNA and Cas9 protein shown in SEQ ID NO.1 or the sgRNA and Cas9 protein shown in SEQ ID NO.2 are transferred into the mouse for fertilization Eggs were subsequently genotyped to obtain C5aR1 knockout model mice.

Further, the transfer is carried out by means of microinjection.

Further, the sgRNA shown in SEQ ID NO.3 and the sgRNA shown in SEQ ID NO.4 are respectively connected to the expression vector for in vitro transcription to obtain sgRNA for microinjection;

The expression vector is a vector carrying a T7 promoter plasmid.

The present invention also provides primer sequences for detecting C5aR1 knockout mice obtained by the above-mentioned method for constructing C5aR1 knockout mice, and the primer sequences are shown in SEQ ID NO. 5-7.

The primers designed in the present invention can only amplify wild-type alleles when the primer sequences are matched with SEQ ID NO. 5-6.

The primer sequences are matched with SEQ ID NO. 5 and 7. This pair of primers is used to confirm the occurrence of gene knockout, and the two primers are designed on both sides of the knockout sequence respectively. In theory, for heterozygous animals, PCR with this pair of primers results in 2 products: the PCR product of the wild-type allele, and the PCR product of the mutant allele; but in practice, the length of these two products is sometimes The difference is very large. The PCR product of the wild-type allele is relatively long, while the PCR product of the mutant allele is relatively short, and the PCR product of the wild-type allele may not be amplified during PCR, so it is impossible to confirm the animal. Specific genotype (including wild-type, heterozygous, homozygous), and the specific position of the mutant gene sequence in the genome and the number of mutant bases cannot be confirmed.

Combined with the results of the above primers, the specific genotype of the animal can be effectively determined: homozygote/heterozygote/wild type.

The present invention also provides a kit for detecting a C5aR1 knockout mouse obtained by the above-mentioned method for constructing a C5aR1 knockout mouse, which contains the above primer sequence.

Further, the kit also includes any one or more of dNTPs, DNA polymerase, buffer, and double distilled water.

The kit provided by the present invention provides convenience for the detection of C5aR1 gene knockout mice.

The present invention also provides a method for detecting a C5aR1 knockout mouse obtained by the above-mentioned method for constructing a C5aR1 knockout mouse. The above-mentioned primer sequence uses the genome of the object to be detected as a template, and performs PCR amplification to obtain the obtained The product is sequenced to determine the genotype of the detection site of the object to be detected.

Further, the object to be detected is a mouse tail genome.

Further, the PCR amplification system is 20-25 μL.

Further, the annealing temperature for PCR amplification was 62°C.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The present invention involves a total of 14 sgRNAs through the analysis of the sequence to be knocked out. After activity detection, these two sgRNAs are obtained. After subsequent experimental verification, they cooperate with the Cas9 protein to obtain a C5aR1 gene knockout mouse.

(2) The present invention also provides a T7-sgRNA for C5aR1 gene knockout, which is ligated into a vector for transcription to obtain a sufficient amount of sgRNA for injection.

(3) The present invention also provides a method for constructing a C5aR1 knockout mouse, which is simple and can effectively obtain a C5aR1 knockout mouse.

(4) The present invention also provides primers, kits and methods for detecting C5aR1 knockout mice, which facilitate the detection of C5aR1 knockout mice.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required to be used in the description of the embodiments or the prior art.

1 is a schematic diagram of the EGE-WFZ-004 gene knockout strategy in Example 1 of the present invention;

Fig. 2 is the activity detection result diagram of Cas9/sgRNA in the embodiment of the present invention 1;

Fig. 3 is the electrophoresis detection diagram of in vitro transcribed sgRNA on T7 promoter plasmid carrier in Example 2 of the present invention;

Fig. 4 is a schematic diagram of identifying primer design in Example 2 of the present invention;

Fig. 5 is the F0 generation genotyping detection result graph in the embodiment of the present invention 2;

Figure 6 is a diagram showing the detection results of F1 generation genotyping EGE-WFZ-004-WT-F/EGE-WFZ-004-Mut-R in Example 2 of the present invention;

Fig. 7 is a graph showing the detection results of F1 generation genotyping EGE-WFZ-004-WT-F/EGE-WFZ-004-WT-R in Example 2 of the present invention.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

Example 1

Design of EGE-WFZ-004 knockout mice

1. Understanding of relevant information and design of targeting strategies

The EGE-WFZ-004 gene is located on the reverse strand of chromosome 7, with a total length of about 12.59kb. Gene ID: 12273. The EGE-WFZ-004 gene has 3 transcripts.

The gene structure of EGE-WFZ-004 was analyzed, and the entire coding region was selected to be deleted. The sgRNAs were designed in the non-conserved regions downstream of Intron1-2 and 3'UTR, respectively, resulting in a genome deletion of approximately 5.5 kb, thus achieving the purpose of EGE-WFZ-004 gene knockout. Model mice were prepared using the EGE system developed by Biositu based on CRISPR/Cas9. The specific knockout strategy is shown in Figure 1.

2. Preparation of EGE-WFZ-004 knockout mice

2.1 Sequencing confirmation of target sequence

Different strains may have different target gene sequences. In order to ensure the efficiency of the designed Cas9/sgRNA, the target site sequence of the C57BL/6 mouse tail needs to be PCR amplified and sequenced to ensure that the sgRNA recognition sequence is completely consistent with the C57BL/6 mouse tail DNA sequence. PCR primers are shown in Table 1.

Table 1 PCR primers

PCR and sequencing of C57BL/6 mouse tail DNA showed that the target sequence of C57BL/6 mouse tail was completely consistent with the sequence given by Genebank and Ensembl.

2.2 Cas9/sgRNA design and activity detection

2.2.1 Design of Cas9/sgRNA

Based on the design principles of sgRNAs, seven sgRNAs were designed at the 5' target site and the 3' target site region, as shown in Table 2 and Table 3.

Table 2 5' target site

Table 3 3' target sites

The sequences of the sgRNAs designed in Tables 2 and 3 were adjusted, and the sequences obtained are 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 GGCCCAATATGTTATACACCA 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 were designed according to Table 4 to synthesize the corresponding primers, which were connected to the pCS-3G vector by Gibson Assembly.

2.3 Activity detection of Cas9/sgRNA

The activity detection of sgRNA was carried out by the CRISPR/Cas9 activity detection method-UCATM method of Biocytometer. It has the advantages of no species restriction, high throughput, wide adaptability, high sensitivity, and simplicity (see the company's website for specific experimental methods).

The obtained detection results are shown in Figure 2.

Comprehensive consideration, EGE-WFZ-004-sgRNA4 and EGE-WFZ-004-sgRNA13 were selected for the next experiment.

Example 2

The EGE-WFZ-004-sgRNA4 and EGE-WFZ-004-sgRNA13 obtained in Example 1 are designed to be connected to a plasmid vector with a T7 promoter for in vitro transcription, 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 were respectively ligated into plasmid vectors with T7 promoter for transcription, and the RNAs obtained are shown in FIG. 3 . The resulting RNA was used for microinjection.

1. Microinjection of Cas9/sgRNA

Cas9/sgRNA were microinjected into mouse (C57BL/6N) fertilized eggs, a total of 332 were injected, and 45 F0 mice were born after injection.

2. Genotype detection of F0 generation mice

The primer design for identification is shown in Figure 4. In Figure 4, arrows represent the positions and orientations of primer design. EGE-WFZ-004-WT-F/EGE-WFZ-004-Mut-R: This pair of primers is used to confirm the occurrence of gene knockout. Two primers are designed on both sides of the knockout sequence. In theory, for heterozygous animals, PCR with this pair of primers will result in 2 products: the PCR product of the wild-type allele, and the PCR product of the mutant allele; but in practice, the lengths of these two products are sometimes The difference is very large. The PCR product of the wild-type allele is relatively long, while the PCR product of the mutant allele is relatively short, and the PCR product of the wild-type allele may not be amplified during PCR, so it is impossible to confirm the animal. Specific genotype (including wild-type, heterozygous, homozygous), and the specific position of the mutant gene sequence in the genome and the number of mutant bases cannot be confirmed.

EGE-WFZ-004-WT-F/EGE-WFZ-004-WT-R: This pair of primers can only amplify the PCR product of the wild-type allele, combined with EGE-WFZ-004-WT-F/EGE- The PCR results of WFZ-004-Mut-R will determine the specific genotype of the animal: homozygous/heterozygous/wild type.

The obtained primers are shown in Table 5.

Table 5 Primer sequences

Note: WT-wild-type allele; Mut-mutant allele.

PCR conditions:

Enzyme:KOD-FX, the system is 20 μl.

Primer: EGE-WFZ-004-WT-F/EGE-WFZ-004-Mut-R, the detection diagram of the obtained PCR product is shown in FIG. 5 .

Through PCR experiments and sequencing results, it is shown 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 is the F0 generation positive mouse.

Due to the rapid cleavage rate in the early embryo, the resulting F0 mice were chimeras. Therefore, the F0 genotype obtained by the identification of the F0 mouse tail is for reference only, and cannot represent that it must be a heritable gene mutation. The heritable genotype needs to be determined after the F1 mouse tail is detected.

3. Genotype identification of F1 generation mice

The above-mentioned positive F0 mice were mated with wild-type mice to obtain F1 generation mice with stable genotypes. The mating results are shown in Table 6.

Table 6 Mating results

Note: "-" means not counted.

The primers used in the detection are the same as above, but the PCR conditions are as follows:

Enzyme: Taq.

The test results are shown in Figures 6 and 7. In Figure 6, Primers: EGE-WFZ-004-WT-F/EGE-WFZ-004-Mut-R; in Figure 7, Primers: EGE-WFZ-004-WT-F/EGE-WFZ-004-WT- R.

After sequencing, the positive genotypes of the F1 generation in Figure 6 are shown in Table 7.

Table 7 F1 generation positive genotyping

The results of PCR identification and sequencing showed that 1E3Z4-006, 1E3Z4-008, 1E3Z4-011, 1E3Z4-016, 1E3Z4-018, and 1E3Z4-020 were F1-positive mice. Among them, 1E3Z4-006 and 1E3Z4-008 have 5468 bases deleted and 3 additional bases inserted; 1E3Z4-011, 1E3Z4-016, 1E3Z4-018, 1E3Z4-020 have 5469 bases deleted and 6 additional inserted. base.

Although specific embodiments of the present invention have been illustrated and described, it should be understood that various other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is intended that all such changes and modifications as fall within the scope of this invention be included in the appended claims.

SEQUENCE LISTING

<110> Peking University Third Hospital

<120> sgRNA, vector, construction method and detection method for C5aR1 gene knockout

<130> 2018

<160> 7

<170> PatentIn version 3.3

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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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