CN110878301A - A sgRNA guide sequence specifically targeting mouse G6pc gene and its application - Google Patents

Abstract

The invention discloses a sgRNA guide sequence of a specific target mouse G6pc gene and application thereof, belonging to the technical field of medical genetics and molecular biology. The nucleotide sequence corresponding to the sgRNA contains a sequence shown in SEQ ID NO.2, and the invention also discloses a method for editing the mouse G6pc gene by using the sgRNA guide sequence of the specific target mouse G6pc gene. The sgRNA guide sequence disclosed by the invention can mediate Cas9 protein, efficiently cuts target DNA, and is further used for editing mouse G6pc gene and influencing the function of mouse G6pc gene encoding protein. The sgRNA guide sequence can realize high-efficiency targeting through a CRISPR/Cas9 system, and the efficiency is 100%.

Description

A sgRNA guide sequence specifically targeting mouse G6pc gene and its application

technical field

The invention relates to a sgRNA guide sequence specifically targeting mouse G6pc gene and its application, belonging to the technical fields of medical genetics and molecular biology.

Background technique

Clustered regularly interspaced short palindromic repeats (Clustered regularly interspaced short palindromic repeats, associated RNA guided endonuclease Cas, CRISPR/Cas) is an acquired immune defense system against foreign genetic material formed in bacteria and archaea during long-term evolution. Among them, the type II CRISPR/Cas9 system has a relatively simple structure and is widely used in gene editing of various species through genetic engineering. The system consists of two elements, Cas9 protein and sgRNA. The sgRNA targets and binds to the complementary sequence of about 20 nt in length of genomic DNA through base complementary pairing, and mediates the cleavage of DNA by the HNH active site and RuvC active site of Cas9 protein. Double strands, resulting in DNA double-strand breaks. Cells repair DNA double-strand breaks by means of non-homologous end joining or homologous recombination. During repair, nucleotides will be randomly inserted or deleted at the break or inserted by homologous recombination, resulting in the repaired sequence and the original sequence. Completely consistent, and then achieve the purpose of gene editing. Compared with ZFNs and TALENs, CRISPR-Cas9 has the advantages of more rapid, simple, efficient, multi-site and specific targeted knockout of genes.

sgRNA, the abbreviation of single guide RNA, the Chinese name is guide RNA. The targeted cleavage of DNA by Cas9 is achieved by the principle of complementary recognition of two small RNAs-crRNA (CRISPR RNA) and tracrRNA (transactivating crRNA) and target sequences. Now two kinds of small RNAs have been fused into one RNA strand, referred to as sgRNA (single guideRNA). Therefore, whether sgRNA can efficiently target target genes is a prerequisite for whether CRISPR-Cas9 can specifically edit target genes, especially for gene knockout and gene knock-in, and the high efficiency of sgRNA is crucial to its impact. Therefore, the ability to design and prepare sgRNAs that efficiently target target genes is the key to gene editing based on the CRISPR-Cas9 system.

The human G6PC gene encodes glucose-6-phosphatase (G6Pase). G6Pase is a phosphatase that hydrolyzes phosphate compounds. It is mainly expressed in liver tissue. It can hydrolyze glucose-6-phosphate into glucose and promote glucose into the blood, thereby regulating blood glucose concentration and maintaining blood glucose balance. It is a key enzyme in glucose metabolism. . Mutations in the G6PC gene can lead to protein dysfunction, causing type I glycogen storage disease. The disease is autosomal recessive and can be affected by both sexes. Clinical symptoms include impaired glucose homeostasis, fasting hypoglycemia, growth retardation, hepatomegaly, nephropathy, hyperlipidemia, hyperuricemia, and lactic acidemia.

G6pc gene mutation is the main cause of human type I glycogen storage disease. There are many types of mutations, including point mutation, synonymous mutation, nonsense mutation and missense mutation. Different mutations cause different clinical symptoms, some of which are different. Disease, some disease, the degree of disease is not the same. Therefore, it is necessary to screen for a clear and typical G6PC pathogenic mutation site, locate the mutation site on the mouse genome, and then target the site through the CRISPR/Cas9 system to establish a G6pc gene mutation cell model Or animal models, so as to accurately simulate type I glycogen storage disease, which is of great significance for in-depth understanding of the pathogenic mechanism of G6PC gene and the exploration of feasible treatment strategies. However, there is currently no sgRNA guide sequence for the mouse G6pc gene and no method for knocking out the gene. In view of this, it is necessary to provide a sgRNA guide sequence that can simulate human pathogenic mutations and specifically target the mouse G6pc gene and a method for using it to specifically knock out the G6pc gene, so as to solve the deficiencies of the existing technology.

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide a sgRNA guide sequence that specifically targets the mouse G6pc gene. The sgRNA guide sequence of the present invention is designed by referring to the pathogenic mutation site of the human G6PC gene and then positioning the site on the mouse G6pc gene according to the DNA sequence of the located site. The sgRNA guide sequence of the present invention can mediate the Cas9 protein to efficiently and specifically cut the target DNA, and then be used to edit the mouse G6pc gene, thereby affecting the function of the protein encoded by the mouse G6pc gene. The sgRNA guide sequence can be efficiently targeted by the CRISPR/Cas9 system with an efficiency of 100%.

The technical solution of the present invention to solve the above problems is as follows: a sgRNA guide sequence that specifically targets the mouse G6pc gene, and the nucleotide sequence corresponding to the sgRNA is the sequence shown in SEQ ID NO.2.

The nucleotide sequence of the sgRNA:

SEQ ID NO. 2: 5'-tgtccaggacccaccaatac-3'.

The inventors of the present application, in order to obtain the above-mentioned sgRNA guide sequence specifically targeting the mouse G6pc gene, carried out the following work:

The first step: The screening of the pathogenic mutation sites of the human G6PC gene was carried out, and the Arg at the 83rd position and the Arg at the 170th position of the human G6PC gene were determined as pseudo-mutation sites.

Type I glycogen storage disease is caused by mutations in the G6PC gene, and 14 pathogenic mutations were screened using the OMIM online database (as shown in Table 1). Due to the limited mutation sites included in OMIM, the present invention also used the ExAC database to screen for inactivating mutations of the human G6PC gene, and a total of 11 mutation sites were screened (as shown in Table 2 and Figure 1). Then use the ClinVar database to search the screened 25 mutation sites to determine the pathogenicity of the mutation (as shown in Figure 2), and finally determine the Arg at position 83 and Arg at position 170 of the human G6PC gene as pseudo-mutations site.

The second step: locate the pathogenic mutation site of the mouse G6PC gene, locate the Arg coding sequence at the 83rd position and the Arg position at the 170th position in the mouse.

Due to the species differences between humans and mice, the protein sequences and functional domains encoded by the same functional gene may be different. Therefore, the inventors of the present application used Clustal Omega online software to compare the protein sequences of human G6PC and mouse G6pc, and found that the human G6PC gene Arg at position 83 and Arg at position 170 correspond to Arg at position 83 and Arg at position 170 in mouse G6pc gene, respectively (as shown in Figure 2). Then, the mouse G6pc gene sequence was derived from the Ensembl database, and the coding sequences of Arg at position 83 and Arg at position 170 were located by Vector NTI software, and then gene targeting sites were designed near the coding sequences.

The third step: gene editing of the pathogenic site: the site that conforms to the characteristics of the 5'-N(21)GG sequence is the editing target of the CRISPR/Cas9 system, and then find the encoding of Arg at position 83 and Arg at position 170 Targets near the sequence.

The gene structure of mouse G6pc is the same as that of human, and its Arg at position 83 and position 170 are encoded by the second and fourth exons, respectively. Use the "Find Motifs" function of Vector NTI software to search for sites with 5'-N(21)GG sequence characteristics, all sites that meet the sequence characteristics are considered to be the editing targets of the CRISPR/Cas9 system, and then find the first Targets near the coding sequences of Arg at position 83 and Arg at position 170 (as shown in Figure 3).

The target sequence of the sgRNA on the G6pc gene conforms to the sequence arrangement rule of 5'-N(21)GG, the target sequence of the sgRNA on the G6pc gene is located in the exon of the gene, and the target sequence of the sgRNA on the G6pc gene The sequences are located on a consensus exon of various spliced forms, and the target sequence of the sgRNA on the G6pc gene is unique.

The second objective of the present invention is to provide a method for editing the mouse G6pc gene using the above-mentioned sgRNA guide sequence specifically targeting the mouse G6pc gene. The present invention utilizes the above-mentioned sgRNA guide sequence specifically targeting the mouse G6pc gene to construct a CRISPR/Cas9 system that can simulate the pathogenic mutation of the human G6PC gene, realizes the efficient transfection of mouse N2a cells, and determines suitable positive cells It can screen the concentration of drugs, and realize the genotype analysis of trace cells, which is used for non-medical diagnosis or treatment purposes.

The technical solution of the present invention to solve the above problems is as follows: a method for editing the mouse G6pc gene using the above-mentioned sgRNA guide sequence specifically targeting the mouse G6pc gene, comprising the following steps:

Step 1: Add accg to the 5' end of the sgRNA guide sequence to synthesize the forward oligonucleotide sequence;

At the same time, the corresponding DNA complementary strand is obtained according to the above-mentioned sgRNA guide sequence, and aaac is added to the 5' end of the DNA complementary strand to synthesize the reverse oligonucleotide sequence;

Annealing the forward oligonucleotide sequence and the reverse oligonucleotide sequence to form double-stranded DNA fragments with cohesive ends;

Step 2: Use Bsa I restriction endonuclease to digest the target vector pGL3-U6-sgRNA plasmid shown in SEQ ID NO.5 to obtain the enzyme-digested product pGL3-U6-sgRNA-Bsa I;

Step 3: Connect the double-stranded DNA fragment with cohesive ends obtained in step 1 and the digestion product pGL3-U6-sgRNA-Bsa I obtained in step 2, transform the ligation product into competent E. coli and coat it on ampicillin-resistant After culturing overnight at 37°C for 20 hours, single clones were picked and the positive clones were identified by sequencing with the universal primer U6 shown in SEQ ID NO. 6. The positive clones were shaken and plasmids were extracted to obtain pGL3-U6- G6pc-sgRNA plasmid;

Step 4: The pGL3-U6-G6pc-sgRNA plasmid obtained in step 3 and the pST1374-NLS-flag-linker-Cas9 expression plasmid shown in SEQ ID NO.7 were co-transfected into mouse N2a cells. After drug screening, Obtain positive sgRNA-Cas9 co-transfected cells;

Step 5: Co-transfect the cells with the positive sgRNA-Cas9 obtained in step 4, carry out cell lysis, and use the cell lysate as a template to carry out PCR amplification reaction of the DNA of the target site, and take the PCR amplification product of the DNA of the target site Sanger sequencing is performed, and if there is a set of peaks at the target site, it is preliminarily confirmed that gene editing has occurred;

Step 6: TA clone sequencing analysis Step 5 preliminarily confirms the genotype of the gene-edited target site, and obtains the gene-edited mouse cells.

On the basis of the above technical solutions, the present invention can also be improved as follows.

Further, in step 1, the annealing reaction system is as follows: 10 μM forward oligonucleotide, 5 μL; 10 μM reverse oligonucleotide, 5 μL; 10×T7 Endonuclease I buffer, 2 μL; ddH ₂ O, 8 μL; The specific reaction program is: 95°C, 5 min; 95°C to 85°C, -1°C/cycle, a total of 10 cycles; 85°C to 25°C, -0.1°C/cycle, a total of 600 cycles, and the annealed product is stored at -20°C .

The further beneficial effects of adopting the above are: by adopting the above reaction system and reaction procedure, more accurate pairing and complementation of the forward oligonucleotide sequence and the reverse oligonucleotide sequence can be realized, thereby efficiently forming double-stranded DNA with sticky ends Fragment. Among them, -1°C/cycle refers to one cycle for every 1°C decrease; -0.1°C/cycle refers to one cycle for every 0.1°C decrease.

Further, in step 2, the reaction system of the restriction enzyme digestion is as follows: pGL3-U6-sgRNA plasmid, 2 μg; 10× digestion buffer, 2 μL; Bsa I restriction enzyme, 2 μL; add ddH ₂ O to the total volume 20 μL, and the digestion reaction system was placed at 37 °C for 3 h.

The above-mentioned further beneficial effects are: using the above-mentioned reaction system, the pGL3-U6-sgRNA plasmid can be fully digested.

Further, in step 3, the specific method for transforming competent E. coli is as follows: take 5 μL of the ligation product and quickly add it to 30 μL of competent E. coli, mix well, stand on ice for 25 min, heat shock in a water bath at 42°C for 90 s, and cool on ice for 90 seconds. Cool for 2 min, add 150 μL of LB liquid medium, rotate at 220 rpm, activate at 37° C. for 30 min, and then coat on the surface of ampicillin-resistant LB solid medium.

The above-mentioned further beneficial effects are: the ligation product can be efficiently transformed into competent Escherichia coli, so that the recombinants are fully activated and the proportion of positive recombinants is increased.

Further, in step 3, in the LB medium with ampicillin resistance, the concentration of ampicillin is 50 μg/mL.

The above-mentioned further beneficial effects are: the use of the LB medium with ampicillin resistance can effectively kill and injure negative Escherichia coli and increase the number of positive colonies.

Further, in step 3, the temperature of the shaking bacteria is 37° C., the rotation speed is 220 rpm, and the culture is performed overnight.

Further, in step 3, the extraction plasmid is extracted using an endotoxin-removing plasmid mid-extraction kit.

The above-mentioned further beneficial effects are: by using the endotoxin-removing plasmid medium extraction kit, high-quality, high-concentration, endotoxin-free plasmids can be obtained, and subsequent cell transfection efficiency is improved.

The above endotoxin-removing plasmid middle extraction kit can be purchased commercially, for example, it can be purchased from Beijing Kangwei Century Biotechnology Co., Ltd., the product number is CW2105S.

Further, in step 4, the drugs are puromycin with a concentration of 50 μg/ml and blasticidin with a concentration of 100 μg/ml.

The above-mentioned further beneficial effects are: using puromycin and blasticidin, the positive transfected cells of sgRNA-Cas9 can be efficiently screened.

Further, in step 4, the mouse N2a cells were inoculated and cultured in DMEM complete medium containing 10% v/v fetal bovine serum before transfection, and cultured in a 37°C, 5% CO ₂ incubator, Fresh medium was replaced every 2d-3d. When the cell confluence reached 80%-90%, it was digested with 0.25% trypsin and passaged, and then divided into 6-well plates. After 18h-20h, the cell confluence reached 60%. -70% for transfection.

The above-mentioned further beneficial effects are: mouse N2a cells, namely mouse neuroma blast cells. Using mouse N2a cells, it can be verified that the sgRNA guide sequence has high gene editing efficiency. Mouse N2a cells have high transfection efficiency of exogenous DNA and high sensitivity to puromycin and blasticidin, which is convenient for drug screening of positive transfected cells.

Mouse N2a cells were transfected using Lipofectamine ^™ 3000 Transfection Reagent (Invitrogen ^™ ) kit. The above-mentioned kits can be purchased commercially, such as from Thermo Fisher Scientific, the product number is L3000015. During transfection, each well (34.8 mm in diameter) was transfected with 2.5 μg of sgRNA expression plasmid and 2.5 μg of Cas9 plasmid.

The above-mentioned culture medium containing fetal bovine serum and DMEM can be purchased from the market, such as from Thermo Fisher Scientific Company, the product number is 16140071 or 11965118, which can achieve the same effect.

Further, in step 5, the PCR amplification reaction system of the target site DNA is: 2 μL of cell lysate, 1 μL of upstream primer, 1 μL of downstream primer, 2 μL of dNTP Mixture, 1 μL of TaKaRa Ex Taq, and 2.5 μL of 10×Ex Taq Buffer , sterilized water was added to 25μL; the PCR amplification reaction program of the target site DNA was: 95°C, 5min; 95°C, 20s, 72°C, 20s, -1°C/cycle, 72°C, 25s, total 10 cycles; 95°C, 20s, 62°C, 25s, 72°C, 25s, a total of 25 cycles; 72°C, 5min, 16°C, infinity.

The above -1°C/cycle refers to one cycle for every 1°C lowering.

Further, the sequence of the upstream primer is shown in SEQ ID NO.8.

SEQ ID NO. 8: 5'-aagaccaccactgcatcgaact-3'.

Further, the sequence of the downstream primer is shown in SEQ ID NO.9.

SEQ ID NO. 9: 5'-caggctgctaggaaggacactc-3'.

The above upstream primers and downstream primers were synthesized by Shanghai Bailiger Biotechnology Co., Ltd. The above-mentioned dNTP Mixture, TaKaRa Ex Taq and 10×Ex Taq Buffer were purchased from Baori Doctor Biotechnology (Beijing) Co., Ltd., the product number is RR001A.

Further, in step 6, the TA cloning and sequencing is specifically: performing gel recovery and purification on the PCR amplification product of the target site obtained in step 5, ligating the purified DNA, and then taking a metal bath at 16°C for 1 h to obtain the ligation product The ligation product was transformed into competent Escherichia coli and spread on LB medium containing ampicillin resistance. After culturing overnight at 37°C for 20 hours, the colony PCR reaction was performed to verify the positive clones, and the positive clones were subjected to Sanger sequencing.

The further beneficial effects of using the above are: using TA cloning and sequencing, a single PCR product can be connected to a carrier, and the genotype of the target site can be obtained through sequencing analysis.

Above-mentioned Solution I and PMD19 carrier can be purchased commercially, such as can be purchased from Bao Ri Doctor Biotechnology (Beijing) Co., Ltd., the article number is 6013.

Further, the reaction system for the connection is: PCR purification product 40ng, Solution I 2.5μL and PMD19 carrier 0.5μL, add water to make up to 5μL; the specific method for the transformation of competent Escherichia coli is: take 5μL of the ligation product and quickly add it to 30 μL of competent Escherichia coli was mixed well, left standing on ice for 25 min, heat-shocked in a water bath at 42 °C for 90 s, cooled on ice for 2 min, added 150 μL of LB liquid medium at a speed of 220 rpm, activated at 37 °C for 30 min, and then Coated on the surface of ampicillin-resistant LB solid medium.

Furthermore, in the LB medium with ampicillin resistance, the concentration of ampicillin is 50 μg/mL.

The further beneficial effect of adopting the above is: adopting the LB medium with ampicillin resistance can effectively kill and injure negative Escherichia coli and increase the number of positive colonies.

Furthermore, the system of the colony PCR reaction is: 1 μL of the colony aqueous solution, 5 μL of Premix Taq enzyme, 0.5 μL of the upstream primer, 0.5 μL of the downstream primer, and supplemented with 10 μL of sterilized water; the procedure of the colony PCR reaction is: 95°C, 5min; 95℃, 20s, 60℃, 20s, 72℃, 25s, 26 cycles; 72℃, 5min.

Further, the sequence of the upstream primer is shown in SEQ ID NO.10.

SEQ ID NO. 10: 5'-gtaaaacgacggccagt-3'.

Further, the sequence of the downstream primer is shown in SEQ ID NO.11.

SEQ ID NO. 11: 5'-caggaaacagctatgac-3'.

The above upstream primers and downstream primers were synthesized by Shanghai Bailiger Biotechnology Co., Ltd. The above-mentioned Premix Taq enzyme was purchased from Bao Ri Doctor Biotechnology (Beijing) Co., Ltd., the item number is R004Q.

Furthermore, the colony aqueous solution is prepared by dissolving monoclonal colonies with a diameter of 1 mm in 10 μL of sterilized water.

Beneficial effects of the present invention:

(1) The sgRNA guide sequence of the present invention can mediate the Cas9 protein to efficiently and specifically cut the target DNA, and then be used to edit the mouse G6pc gene, thereby affecting the function of the protein encoded by the mouse G6pc gene. The sgRNA guide sequence can be efficiently targeted by the CRISPR/Cas9 system with an efficiency of 100%.

(2) The present invention utilizes the above-mentioned sgRNA guide sequence specifically targeting the mouse G6pc gene to construct a CRISPR/Cas9 system that can simulate the pathogenic mutation of the human G6PC gene, realizes the efficient transfection of mouse N2a cells, and determines the appropriate The concentration of positive cells for drug screening, and the genotype analysis of trace cells is realized, which is used for non-medical diagnosis or treatment purposes. effect.

Description of drawings

Figure 1 is a schematic diagram of the exon structure and function-inactivating mutations of the human G6PC gene in the ExAC database. In the figure, the dots represent inactivating mutations, and the arrows represent the direction of transcription.

Figure 2 is an alignment of human and mouse G6PC protein sequences. The first box from top to bottom is the 83rd Arg of human and mouse to be mutated; the second box from top to bottom is the 170th Arg of human and mouse.

Figure 3 shows the gene editing design and target sequence of the mouse G6pc pathogenic locus.

Figure 4 is a PCR verification diagram of positive clones. In the figure, M represents Marker, 1 and 2 represent pGL3-U6-G6pc-sgRNA1; 3 and 4 represent pGL3-U6-G6pc-sgRNA2; 5 and 6 represent pGL3-U6-G6pc-sgRNA3; 7 and 8 represent pGL3-U6 -G6pc-sgRNA4.

Figure 5 is the sgRNA1 target sequence. In the figure, the black shading is the sgRNA1 wild-type target sequence.

Figure 6 shows the sgRNA2 target gene editing sequence.

Figure 7 is the sgRNA3 target sequence. In the figure, the black shading is the sgRNA3 wild-type target sequence.

Figure 8 is the sgRNA4 target sequence. In the figure, the black shading is the sgRNA4 wild-type target sequence.

Figure 9 is the sgRNA2 target sequence. In the figure, the black shading is the sgRNA2 wild-type target sequence.

Detailed ways

The principles and features of the present invention will be described below with reference to the specific drawings. The examples are only used to explain the present invention, but not to limit the scope of the present invention.

1. Screening of pathogenic mutation sites of human G6PC gene to determine Arg at position 83 and Arg at position 170 of human G6PC gene as pseudo-mutation sites

Type I glycogen storage disease is caused by mutations in the G6PC gene, and 14 pathogenic mutations were screened using the OMIM online database (as shown in Table 1). Due to the limited mutation sites included in OMIM, the present invention also used the ExAC database to screen for inactivating mutations of the human G6PC gene, and a total of 11 mutation sites were screened (as shown in Table 2 and Figure 1). Then use the ClinVar database to search the screened 25 mutation sites to determine the pathogenicity of the mutation (as shown in Figure 2), and finally determine the Arg at position 83 and Arg at position 170 of the human G6PC gene as pseudo-mutations site.

Table 1 Types of pathogenic mutations in human G6PC gene in the OMIM database

Table 2 Inactivating mutations of human G6PC in the ExAC database

The ClinVar database was searched, and (G6PC): c.247C>T (p.Arg83Cys) was identified as the pathogenic variant of glycogenosis type 1A, accession number: RCV000012778.10.

The ClinVar database was searched, and (G6PC): c.508C>T (p.Arg170Ter) was identified as the pathogenic mutation of glycogen storage disease type 1A, accession number: RCV000624988.3.

2. The location of the pathogenic mutation site of the mouse G6PC gene, the coding sequence of Arg at position 83 and Arg at position 170 in mouse.

Due to the species differences between humans and mice, the protein sequences and functional domains encoded by the same functional gene may be different. Therefore, the inventors of the present application used Clustal Omega online software to compare the protein sequences of human G6PC and mouse G6pc, and found that the human G6PC gene Arg at position 83 and Arg at position 170 correspond to Arg at position 83 and Arg at position 170 in mouse G6pc gene, respectively (as shown in Figure 2). Then the mouse G6pc gene sequence was derived from the Ensembl database, and the coding sequences of Arg at position 83 and Arg at position 170 were located by Vector NTI software, and then gene editing sites were designed near the coding sequences.

3. Gene editing of pathogenic loci: The locus that conforms to the characteristics of the 5'-N(21)GG sequence is the editing target of the CRISPR/Cas9 system, and then find the 83rd position Arg and the 170th position near the coding sequence of Arg target

The gene structure of mouse G6pc is the same as that of human, and its Arg at position 83 and position 170 are encoded by the second and fourth exons, respectively. Use the "Find Motifs" function of Vector NTI software to search for sites with 5'-N(21)GG sequence characteristics, all sites that meet the sequence characteristics are considered to be the editing targets of the CRISPR/Cas9 system, and then find the first Targets near the coding sequences of Arg at position 83 and Arg at position 170 (as shown in Figure 3).

The target sequence of sgRNA on G6pc gene conforms to the sequence arrangement rule of 5'-N(21)GG, the target sequence of sgRNA on G6pc gene is located in the exon of the gene, and the target sequence of sgRNA on G6pc gene is located in different The target sequence of the sgRNA on the G6pc gene is unique on the consensus exon in the spliced form.

The present invention designs a total of 4 sgRNA guide sequences for targeting sites, which are respectively the sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4.

SEQ ID NO. 1: 5'-tgtttggacaacgcccgtat-3' (hereinafter referred to as "sgRNA1").

SEQ ID NO. 2: 5'-tgtccaggacccaccaatac-3' (hereinafter referred to as "sgRNA2").

SEQ ID NO. 3: 5'-agctgaacgtctgtctgtcc-3' (hereinafter referred to as "sgRNA3").

SEQ ID NO. 4: 5'-gtgagcagcaaggtagatcc-3' (hereinafter referred to as "sgRNA4").

4. Editing the mouse G6pc gene using the above sgRNA guide sequences

Step 1: Construction of sgRNA expression vector

The forward oligonucleotide sequences (G6pc-M-sg1 ⁺ , G6pc-M-sg2 ^{+ , G6pc-M-sg3 + , G6pc-M-sg4 + , G6pc-M-sg4 + , G6pc-M-sg4 + , G6pc-M-sg4 +} , G6pc-M-sg4 ⁺ , G6pc-M-sg4 ⁺ );

At the same time, the corresponding DNA complementary strand is obtained according to the above-mentioned sgRNA guide sequence, and the reverse oligonucleotide sequence (G6pc-M-sg1-, G6pc ^- M-sg2-, G6pc-M-sg2-, G6pc-M-sg2-, G6pc-M-sg2-, G6pc-M ^- sg2-, G6pc-M ^- sg3-, G6pc-M ^- sg4-);

The forward and reverse oligonucleotide sequences are annealed to form double-stranded DNA fragments with cohesive ends. The annealing reaction system is: 10 μM forward oligonucleotide, 5 μL; 10 μM reverse oligonucleotide, 5 μL; 10×T7Endonuclease I buffer, 2 μL; ddH ₂ O, 8 μL; the reaction program is: 95°C , 5min; 95°C to 85°C, -1°C/cycle (that is, for every 1°C decrease, cycle once), a total of 10 cycles; 85°C to 12°C, -0.1°C/cycle (that is, for every 0.1°C decrease, cycle once) ) for a total of 600 cycles, and the annealed products were stored at -20°C.

Chemically synthesized forward oligonucleotides and reverse oligonucleotides are shown in Table 3.

Table 3 Chemically synthesized forward oligonucleotides and reverse oligonucleotides

G6pc-M-sg1⁺ 5'-accgtgtttggacaacgcccgtat-3' (SEQ ID NO. 14) G6pc-M-sg1^- 5'-aaacatacgggcgttgtccaaaca-3' (SEQ ID NO. 15) G6pc-M-sg2⁺ 5'-accgtgtccaggacccaccaatac-3' (SEQ ID NO. 16) G6pc-M-sg2^- 5'-aaacgtattggtgggtcctggaca-3' (SEQ ID NO. 17) G6pc-M-sg3⁺ 5'-accgagctgaacgtctgtctgtcc-3' (SEQ ID NO. 18) G6pc-M-sg3^- 5'-aaacggacagacagacgttcagct-3' (SEQ ID NO. 19) G6pc-M-sg4⁺ 5'-accggtgagcagcaaggtagatcc-3' (SEQ ID NO. 20) G6pc-M-sg4^- 5'-aaacggatctaccttgctgctcac-3' (SEQ ID NO. 21)

Step 2: The target vector pGL3-U6-sgRNA plasmid shown in SEQ ID NO. 5 was digested with Bsa I restriction endonuclease to obtain the digested product pGL3-U6-sgRNA-Bsa I. Among them, the reaction system of restriction enzyme digestion is as follows: pGL3-U6-sgRNA plasmid, 2 μg; 10× digestion buffer, 2 μL; Bsa I restriction endonuclease, 2 μL; supplemented with ddH ₂ O to a total volume of 20 μL, and the restriction enzyme digestion reaction was carried out. The system was placed at 37°C to react for 3h.

Step 3: Connect the double-stranded DNA fragments with sticky ends obtained in step 1 to the digestion product pGL3-U6-sgRNA-Bsa I obtained in step 2, respectively, take 5 μL of the ligation product and quickly add it to 30 μL of competent E. coli. Mix well, let stand on ice for 25 min, heat shock in 42 °C water bath for 90 s, cool on ice for 2 min, add 150 μL of LB liquid medium, rotate at 220 rpm, and activate at 37 °C for 30 min. Then, it was coated on the surface of ampicillin-resistant LB solid medium (ampicillin concentration was 50 μg/mL), and after culturing overnight at 37°C for 20 h, single clones were picked and positive by bacterial liquid PCR and agarose gel electrophoresis clone (as shown in Figure 4). The positive clones were incubated overnight at 37°C with a rotating speed of 220 rpm. Plasmids were extracted with endotoxin-removing plasmid mid-extraction kit to obtain pGL3-U6-G6pc-sgRNA1 plasmid, pGL3-U6-G6pc-sgRNA2 plasmid, pGL3-U6-G6pc-sgRNA3 plasmid and pGL3-U6-G6pc-sgRNA4 plasmid, respectively. The above plasmid is sequenced using the universal primer U6 shown in SEQ ID NO. 6 to confirm that the target DNA fragment is inserted into the specific site of the vector. The above endotoxin-removing plasmid middle extraction kit can be purchased commercially, for example, it can be purchased from Beijing Kangwei Century Biotechnology Co., Ltd., the product number is CW2105S.

SEQ ID NO. 6: 5'-atggactatcatatgcttaccgta-3'.

Step 4: The mouse N2a cells were first inoculated and cultured in DMEM complete medium containing 10% v/v fetal bovine serum, and cultured in a 37°C, 5% CO ₂ incubator, with fresh medium replaced every 2d-3d, After the cells reached 80%-90% confluence, they were digested with 0.25% trypsin and passaged. Then, it was divided into 6-well plates, and after 18h-20h, the cells were transfected when the confluence reached 60%-70%.

Lipofectamine ^TM 3000 Transfection Reagent (Invitrogen ^TM ) kit was used to load pGL3-U6-G6pc-sgRNA1 plasmid, pGL3-U6-G6pc-sgRNA2 plasmid, pGL3-U6-G6pc-sgRNA3 plasmid, pGL3-U6-G6pc- The sgRNA4 plasmid, and the pST1374-NLS-flag-linker-Cas9 expression plasmid shown in SEQ ID NO. 7, were co-transfected into mouse N2a cells. The above-mentioned Lipofectamine ^TM 3000 Transfection Reagent (Invitrogen ^TM ) kit can be purchased commercially, for example, can be purchased from Thermo Fisher Scientific, the product number is L3000015. During transfection, each well (34.8 mm in diameter) was transfected with 2.5 μg of sgRNA expression plasmid and 2.5 μg of pST1374-NLS-flag-linker-Cas9 expression plasmid.

Puromycin at a concentration of 50 μg/ml and blasticidin at a concentration of 100 μg/ml were used for drug screening, and positive sgRNA1-Cas9 co-transfected cells, sgRNA2-Cas9 co-transfected cells, and sgRNA3-Cas9 co-transfected cells were obtained, respectively. Transfected cells and sgRNA4-Cas9 co-transfected cells. The above four groups of co-transfected cells were washed three times with phosphate buffer, then digested with 0.25% trypsin, and collected by centrifugation.

Step 5: Co-transfect cells with positive sgRNA1-Cas9, positive sgRNA2-Cas9, positive sgRNA3-Cas9 and positive sgRNA4- Cas9 co-transfected cells were centrifuged at 8000 rpm for 5 min to remove the supernatant, and 8 μL of AD1 was added to suspend the cell pellet, then 8 μL of the liquid was transferred to a PCR tube, 2 μL of AD2 was added, and incubated at 55°C for 10 min, 95°C for 3 min ; Add 8 μL of AD3 and mix well to obtain cell lysates of 4 groups of cells, which are used as templates for subsequent PCR amplification of target site DNA, and stored at -20°C. The above-mentioned TransDirect Animal Tissue PCR kit can be purchased commercially, for example, it can be purchased from Beijing Quanshijin Biological Co., Ltd., the product number is AD201-01.

The cell lysates of the above four groups of cells were used as templates to carry out the PCR amplification reaction of the target site DNA. Among them, the PCR amplification reaction system of the target site DNA of sgRNA1 and sgRNA2 is: cell lysate 2 μL, upstream primer 1 μL, downstream primer 1 μL, dNTP Mixture 2 μL, TaKaRa Ex Taq 1 μL, 10×Ex Taq Buffer 2.5 μL, sterilization Bacteria water was added to 25 μL. The sequence of the upstream primer is shown in SEQ ID NO. 8, SEQ ID NO. 8: 5'-aagaccaccactgcatcgaact-3'. The sequence of the downstream primer is shown in SEQ ID NO.9, SEQ ID NO.9:

5'-caggctgctaggaaggacactc-3'.

The PCR amplification reaction system of the target site DNA of sgRNA3 and sgRNA4 is: 2 μL of cell lysate, 1 μL of upstream primer, 1 μL of downstream primer, 2 μL of dNTP Mixture, 1 μL of TaKaRa Ex Taq, 2.5 μL of 10×Ex Taq Buffer, sterilized water Make up to 25 μL. The sequence of the upstream primer is shown in SEQ ID NO. 12, SEQ ID NO. 12: 5'-gagaccctaactggagaccaag-3'. The sequence of the downstream primer is shown in SEQ ID NO.13, SEQ ID NO.13:

5'-ctggaatacaggcaggtaaatc-3'.

The above two sets of upstream primers and downstream primers were synthesized by Shanghai Bailiger Biotechnology Co., Ltd. The above-mentioned dNTP Mixture, TaKaRa Ex Taq and 10×Ex Taq Buffer were purchased from Baori Doctor Biotechnology (Beijing) Co., Ltd., the product number is RR001A.

The above four groups of PCR amplification reactions of target site DNA were as follows: 95°C, 5min; 95°C, 20s, 72°C, 20s, -1°C/cycle (that is, every 1°C decrease, cycle once), 72°C , 25s, a total of 10 cycles; 95°C, 20s, 62°C, 20s, 72°C, 25s, a total of 25 cycles; 72°C, 5min, 16°C, infinite. 5 μL of PCR amplification products of the above-mentioned 4 groups of target site DNA were respectively taken and detected by agarose gel electrophoresis with a mass percentage of 1%, and mouse genomic DNA was used as a control.

The PCR products of the above-mentioned 4 groups of target site DNA were respectively taken for Sanger sequencing, and Sanger sequencing was completed by Shanghai Bailiger Biotechnology Co., Ltd. If a set of peaks appears at the target site, it is preliminarily confirmed that gene editing has occurred. The sequencing results are shown in Figures 5-8. The sequencing results showed that among the four designed targeting sites, compared with the wild-type targeting sites (as shown in Figure 9), only the sgRNA2 target underwent gene editing (as shown in Figure 6), proving that only sgRNA2 Sites are reliable gene editing targets, and other targets cannot be used for gene editing.

Step 6: TA clone sequencing analysis Step 5 preliminarily confirms the genotype of the gene-edited target site, and obtains the gene-edited mouse cells.

The PCR product of the amplified sgRNA2 target was recovered and purified by gel, and the purified DNA was ligated. The ligation reaction system was: 40 ng of the purified PCR product, 2.5 μL of Solution I and 0.5 μL of the PMD19 vector, supplemented with water to 5 μL; then 16 ℃ metal bath for 1 h to obtain the ligated product. Above-mentioned Solution I and PMD19 carrier can be purchased commercially, such as can be purchased from Bao Ri Doctor Biotechnology (Beijing) Co., Ltd., the article number is 6013.

Quickly add 5 μL of ligation product to 30 μL of competent E. coli, mix well, let stand on ice for 25 min, heat shock in 42°C water bath for 90 s, cool on ice for 2 min, add 150 μL of LB liquid medium, and rotate at 220 rpm , activated at 37°C for 30min. Then, it was coated on the surface of ampicillin-resistant LB solid medium (ampicillin concentration was 50 μg/mL), and after culturing overnight at 37° C. for 20 h, the colony PCR reaction was verified. The system of colony PCR reaction was: 1 μL of colony aqueous solution, 5 μL of PremixTaq enzyme, 0.5 μL of upstream primer, 0.5 μL of downstream primer, and sterilized water supplemented to 10 μL. Wherein, the colony aqueous solution is prepared by dissolving monoclonal colonies with a diameter of 1 mm in 10 μL of sterilized water. The sequence of the upstream primer is shown in SEQ ID NO.10, SEQ ID NO.10:

5'-gtaaaacgacggccagt-3'. The sequence of the downstream primer is shown in SEQ ID NO. 11, SEQ ID NO. 11: 5'-caggaaacagctatgac-3'.

The above upstream primers and downstream primers were synthesized by Shanghai Bailiger Biotechnology Co., Ltd. The above-mentioned Premix Taq enzyme was purchased from Bao Ri Doctor Biotechnology (Beijing) Co., Ltd., the item number is R004Q.

The procedure of colony PCR reaction was: 95°C, 5min; 95°C, 20s, 60°C, 20s, 72°C, 25s, 26 cycles; 72°C, 5min, and then electrophoresis identification.

Positive clones were screened and subjected to Sanger sequencing. Sanger sequencing was performed by Shanghai Bailiger Biotechnology Co., Ltd. The alignment analysis between the sequencing results and the original sequence showed that random insertion and deletion of bases occurred at the sg2 target site, and the editing efficiency was 100% (as shown in Table 4).

Table 4 Genotype analysis of sgRNA2 targets

Note: Bold fonts represent target sequences; italic letters represent insertion sequences.

It can be seen that the sgRNA2 guide sequence can mediate the Cas9 protein to efficiently and specifically cut the target DNA, and then be used to knock out the mouse G6pc gene and eliminate the expression of the mouse G6pc gene. The sgRNA guide sequence can be efficiently targeted by the CRISPR/Cas9 system with an efficiency of 100%.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. within the range.

sequence listing

<110> Guilin Medical College

<120> A kind of sgRNA guide sequence specifically targeting mouse G6pc gene and its application

<160> 31

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 1

tgtttggaca acgcccgtat 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 2

tgtccaggac ccaccaatac 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 3

agctgaacgt ctgtctgtcc 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 4

gtgagcagca aggtagatcc 20

<210> 5

<211> 4951

<212> DNA

<213> Artificial Sequence

<400> 5

ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg 60

cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 120

ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 180

cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 240

tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 300

tgtggaaagg acgaaacacc gggagaccga gagagggtct cagttttaga gctagaaata 360

gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt 420

tttttaaaga attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa 480

aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca 540

tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca 600

gggacagcag agatccactt tggccgcggc tcgagggggt tggggttgcg ccttttccaa 660

ggcagccctg ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc 720

ggcgccgacc ctgggactcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc 780

gccgctaccc ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt 840

tccttgcggt tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc 900

ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc 960

aatagcggct gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag 1020

gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc 1080

aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct 1140

ccccaggggg atccaccgga gcttaccatg accgagtaca agcccacggt gcgcctcgcc 1200

acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc 1260

gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa 1320

ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc 1380

gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc 1440

ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc 1500

ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg 1560

cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc 1620

gagcgcgccg gggtgcccgc cttcctggaa acctccgcgc cccgcaacct ccccttctac 1680

gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg 1740

tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa 1800

aggagcgcac gaccccatgc atcggtacct ttaagaccaa tgacttacaa ggcagctgta 1860

gatcttagcc actttctaga gtcggggcgg ccggccgctt cgagcagaca tgataagata 1920

cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga 1980

aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa 2040

caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag 2100

caagtaaaac ctctacaaat gtggtaaaat cgataaggat ccgtcgaccg atgcccttga 2160

gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac 2220

ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctcttccgct 2280

tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 2340

tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 2400

gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 2460

aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 2520

ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 2580

gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 2640

ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 2700

ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 2760

cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 2820

attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 2880

ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 2940

aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggtttttttt 3000

gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 3060

tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 3120

ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 3180

taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 3240

atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 3300

actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgggaccca 3360

cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 3420

agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 3480

gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 3540

gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 3600

gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 3660

gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 3720

cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 3780

ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 3840

accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 3900

aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 3960

aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 4020

caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 4080

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 4140

gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 4200

cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 4260

accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 4320

gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 4380

tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 4440

gggccatcgc cctgatagac ggttttttcgc cctttgacgt tggagtccac gttctttaat 4500

agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 4560

ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 4620

tttaacgcga attttaacaa aatattaacg tttacaattt cccattcgcc attcaggctg 4680

cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gcccaagcta 4740

ccatgataag taagtaatat taaggtacgg gaggtacttg gagcggccgc aataaaatat 4800

ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta ctaacatacg 4860

ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc cccagtgcaa 4920

gtgcaggtgc cagaacattt ctctatcgat a 4951

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 6

atggactatc atatgcttac cgta 24

<210> 7

<211> 9317

<212> DNA

<213> Artificial Sequence

<400> 7

agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60

acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120

tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180

ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct 240

agctagaggt cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac 300

aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 360

tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat 420

ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt 480

catagaaggc ggcggtggaa tcgaaatctc gtagcacgtg tcagtcctgc tcctcggcca 540

cgaagtgctt agccctccca cacataacca gagggcagca attcacgaat cccaactgcc 600

gtcggctgtc catcactgtc cttcactatg gctttgatcc caggatgcag atcgagaagc 660

acctgtcggc accgtccgca ggggctcaag atgcccctgt tctcatttcc gatcgcgacg 720

atacaagtca ggttgccagc tgccgcagca gcagcagtgc ccagcaccac gagttctgca 780

caaggtcccc cagtaaaatg atatacattg acaccagtga agatgcggcc gtcgctagag 840

agagctgcgc tggcgacgct gtagtcttca gagatgggga tgctgttgat tgtagccgtt 900

gctctttcaa tgagggtgga ttcttcttga gacaaaggct tggccatggt ttagttcctc 960

accttgtcgt attatactat gccgatatac tatgccgatg attaattgtc aacacgtgct 1020

gatcagatcc gaaaatggat atacaagctc ccgggagctt tttgcaaaag cctaggcctc 1080

caaaaaagcc tcctcactac ttctggaata gctcagaggc agaggcggcc tcggcctctg 1140

cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta 1200

ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc 1260

tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat 1320

tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta 1380

actgacacac attccacaga attaattcgc gttaaatttt tgttaaatca gctcattttt 1440

taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg 1500

gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt 1560

caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc 1620

aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg 1680

atttagagct tgacgggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa 1740

aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc 1800

cgccgcgctt aatgcgccgc tacagggcgc gtggggatac cccctagagc cccagctggt 1860

tctttccgcc tcagaagcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc 1920

ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact 1980

cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc 2040

cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac 2100

agtcgaggct gatcagcggg tttaaactca atggtgatgg tgatgatgac cggtacgcgt 2160

agaatcgaga ccgaggagag ggttagggat aggcttacct tcgaagggcc cctagtcgcc 2220

gccgagctga gacaggtcga tgcgagtctc gtacagtccg gtaattgact ggtgtatcag 2280

ggtggcatcg agaacttctt tggtagaggt gtaccgcttc ctgtcaatag ttgtatcgaa 2340

atacttgaag gcagcaggag cgcccagatt agtcagagta aagaggtgga taatattctc 2400

tgcttgttcg cgaattggct tgtccctgtg cttattatat gcgctcagca ctttatcgag 2460

gtttgcatcg gccagaataa cccgcttgct gaactcgcta atctgttcaa tgatttcgtc 2520

caggtagtgt ttatgttgct caacaaagag ttgcttctgc tcattgtctt cagggctacc 2580

tttgagtttc tcgtagtggg aggccagata caggaagttc acgtatttgg agggcagagc 2640

cagctcgttt cctttctgca gctctccggc ggaggccagc atccgcttcc taccattctc 2700

cagctcaaag agagagtact tgggcagttt gatgatgaga tctttcttca cttctttata 2760

gcccttagct tccaggaaat cgattggatt cttctcgaag ctggatctct ccataatagt 2820

aattccgagc agctccttaa cagacttgag tttcttggac ttgcctttct ccacttttgc 2880

cacgaccaga acggaataag ccactgtagg ggaatcgaaa ccgccatact tctttgggtc 2940

ccaatctttc ttcctggcga tcagcttgtc agagttccgc tttggcagga tgctctcctt 3000

tgagaatccg ccggtctgca cttcggtctt cttcacgata ttgacttgtg gcatggacag 3060

caccttccgc acagttgcga agtccctgcc tttatcccac acgatttctc ctgtttctcc 3120

gtttgtttcg atcagtgggc gcttccggat ttcgccgtta gccagggtta tctcagtctt 3180

aaagaaattc atgatattag agtagaagaa gtacttggcg gtggctttgc caatctcttg 3240

ctcagacttt gctatcatct tcctcacatc gtagacttta tagtcaccgt acacgaactc 3300

agactccagt ttagggtatt tcttgatcag ggcggtgcca acgacagcat tgagataggc 3360

atcgtgagca tgatggtaat tgtttatttc gcgaactttg tagaattgaa agtccttccg 3420

gaagtcagac accagcttgc tcttcagagt tatcactttc acttccctga tcagcttatc 3480

gttctcatcg tacttagtgt tcatcctaga gtcgaggatt tgtgccacgt gtttggtaat 3540

ctggcgtgtt tcgaccagtt gcctcttaat aaagccggcc ttgtcgagtt cgctcagacc 3600

tcctctttct gccttagtca gattgtcaaa cttccgctgg gtgatcagtt tggcgttgag 3660

gagctgccgc cagtaattct tcatcttctt gaccacctct tctgatggaa cattgtcaga 3720

cttaccgcga ttcttatcgg atctggtcag caccttgttg tcaatggagt catctttgag 3780

gaaggactgt ggaacaatat ggtccacgtc ataatcggac agccggttga tgtcgagttc 3840

ctggtcaacg tacatgtccc gcccgttctg gaggtagtac aggtagagtt tctcgttctg 3900

gagctgtgta ttctccacag ggtgctcctt cagtatctga gatcccagct ccttaattcc 3960

ctcttcgatt cttttcatcc gttcccgaga gttcttctgg cccttctggg tggtttggtt 4020

ctcccttgcc atttcgataa cgatattctc tggcttgtgc cgacccatca ctttgacgag 4080

ttcgtccacg accttgactg tctgcagtat tcccttcttt atggcgggtg atccagcgag 4140

gttggcgatg tgctcgtgca ggctgtcgcc ttgaccgctc acctgtgcct tctggatgtc 4200

ctctttaaat gtcagagagt catcgtgaat cagttgcatg aagttcctgt tagcgaatcc 4260

gtcggatttc aggaaatcca ggatggtctt tccgctctgt ttgtcgcgga tgccgtttat 4320

gagtttcctg gagagtctac cccacccagt gtagcgccgc cgcttgagct gtttcatgac 4380

tttatcgtca aacagatggg cataggtctt caggcgctct tcgatcatct ctctatcctc 4440

gaacagagtc agggtcagca cgatatcttc caggatgtcc tcgttctcct cattatcgag 4500

gaaatctttg tctttgatta tcttcagcag atcatggtaa gtgcccaggc tggcattaaa 4560

gcggtcctcc acgccagaga tttccactga gtcaaagcat tcgatcttct taaagtaatc 4620

ctccttgagc tgcttcactg tcacctttct attagtcttg aagagcagat caacgatagc 4680

cttcttctgc tctccggaca ggaaggcagg cttcctcatg ccctcggtca cgtacttcac 4740

tttagtcagc tcgttataga cggtgaaata ctcgtacagg agtgaatgtt tgggcaggac 4800

cttctcgttg ggcaggttct tatcgaaatt ggtcatccgt tcgatgaatg actgggcgct 4860

tgcgccctta tccacgacct cttcgaagtt ccaaggagta attgtctcct cagatttcct 4920

ggtcatccaa gcgaagcggg agttgcctct agccagaggg ccgacgtaat aagggatcct 4980

gaaggtcagg atcttctcta tcttctcccg gttatccttc aggaaaggat agaaatcctc 5040

ctgcctgcgg aggattgcat gcagctctcc caggtgtatc tgatgtggaa tggagccatt 5100

atcaaaggtc ctctgcttcc tcagcaggtc ctccctgttc agcttcacca gcagctcttc 5160

agtaccgtcc atcttctcga ggattggttt gatgaacttg taaaattctt cctgtgatgc 5220

tccgccatcg atgtatccgg catatccatt cttgctctgg tcgaagaata tctctttgta 5280

cttctctggc agctgttgcc tcacgagggc tttgagcaga gtcaggtctt gatggtgttc 5340

atcatagcgt tttatcatgg aggcgctcag aggtgctttg gtgatctcag tgttgacccg 5400

gagtatgtcg ctcagcagaa tggcgtcgga gagattctta gcagccagaa agagatcggc 5460

gtactggtcg cctatctgtg cgagcaggtt gtccagatca tcgtcatagg tgtccttgga 5520

gagctggagt ttagcatctt cggccagatc aaaattggac ttgaagttag gtgtcaggcc 5580

caggctcagg gcgatgaggt tcccaaacag gccgttcttc ttttctcctg gcagttgggc 5640

aatcagattc tccagtctgc gtgacttgga cagccgagcg gacagaatag ccttggcatc 5700

cacaccagaa gcgtttatgg gattctcctc gaacagttgg ttatatgtct gcaccagttg 5760

aatgaagagt ttatccacat cggaattatc gggattcagg tcgccctcga tcagaaagtg 5820

tcctctaaac tttatcatat gagccagggc cagatagatg agcctcaggt ctgctttatc 5880

ggtgctgtcc accagcttct tcctcagatg gtagattgta ggatactttt catggtaagc 5940

cacctcatcc acgatatttc cgaatatagg gtgcctctcg tgtttcttat cctcctccac 6000

cagaaagctc tcttccaggc ggtgaaagaa ggagtcgtcc accttagcca tttcgttgct 6060

aaagatctct tgcagataac atatccgatt cttgcggcgg gtgtatctcc gccttgcggt 6120

ccgcttcagc cgagtagctt cagcggtttc accggagtcg aagaggagtg ctccgatcag 6180

gttcttcttg attgaatggc ggtcagtatt acccagcacc ttgaatttct tgcttggcac 6240

cttatactcg tcggttatga cggcccagcc aacggagttt gtcccgatat ccagtccaat 6300

agagtatttc ttgtctctag tgtgggtccg ctggtgtctg gtcagagcac cagactgaga 6360

gaaagattta ccacactctg ggcatttgta tggcttctcg ccgggttcca gtctagattt 6420

atcgtcgtca tccttgtagt cagcggccgc caccttcctc tttttcttag gtcccatggt 6480

gctagccagc ttgggtctcc ctatagtgag tcgtattaat ttcgataagc cagtaagcag 6540

tgggttctct agttagccag agagctctgc ttatatagac ctcccaccgt acacgcctac 6600

cgcccatttg cgtcaatggg gcggagttgt tacgacattt tggaaagtcc cgttgatttt 6660

ggtgccaaaa caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt 6720

caaaccgcta tccacgccca ttgatgtact gccaaaaccg catcaccatg gtaatagcga 6780

tgactaatac gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat 6840

aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat 6900

acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat 6960

ggaaagtccc tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg 7020

ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca 7080

tatatgggct atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc 7140

aatgtcaacg cgtatatctg gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa 7200

gcagattctt catgcaattg tcggtcaagc cttgccttgt tgtagcttaa attttgctcg 7260

cgcactactc agcgacctcc aacacacaag cagggagcag atactggctt aactatgcgg 7320

catcagagca gattgtactg agagtgcacc ataggggatc gggagatctc ccgatccgtc 7380

gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 7440

aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 7500

ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 7560

ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 7620

agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 7680

tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 7740

tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 7800

ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 7860

gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 7920

acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 7980

tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 8040

gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 8100

actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 8160

aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 8220

cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 8280

tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 8340

cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 8400

tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 8460

ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 8520

ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 8580

cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 8640

aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 8700

agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 8760

tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 8820

ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 8880

cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 8940

atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 9000

ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 9060

tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 9120

gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 9180

gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 9240

cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 9300

gagcgaggaa gcggaag 9317

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 8

aagaccacca ctgcatcgaa ct 22

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 9

caggctgcta ggaaggacac tc 22

<210> 10

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 10

gtaaaacgac ggccagt 17

<210> 11

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 11

caggaaacag ctatgac 17

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 12

gagaccctaa ctggagacca ag 22

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 13

ctggaataca ggcaggtaaa tc 22

<210> 14

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 14

accgtgtttg gacaacgccc gtat 24

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 15

aaacatacgg gcgttgtcca aaca 24

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 16

accgtgtcca ggacccacca atac 24

<210> 17

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 17

aaacgtattg gtgggtcctg gaca 24

<210> 18

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 18

accgagctga acgtctgtct gtcc 24

<210> 19

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 19

aaacggacag acagacgttc agct 24

<210> 20

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 20

accggtgagc agcaaggtag atcc 24

<210> 21

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 21

aaacggatct accttgctgc tcac 24

<210> 22

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 22

ttggacaacg cccgtattgg tgggtcctgg acaccgacta cta 43

<210> 23

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 23

ttggacaacg cccgtattag gtgggtcctg gacaccgact acta 44

<210> 24

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 24

ttggacaacg cccgtagtgg gtcctggaca ccgactacta 40

<210> 25

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 25

ttggacaacg cccgtttggt gggtcctgga caccgactac ta 42

<210> 26

<211> 57

<212> DNA

<213> Artificial Sequence

<400> 26

ttggacaacg cccgtattgg tgggtacccc accctacccc ccacccacag ttacaag 57

<210> 27

<211> 57

<212> DNA

<213> Artificial Sequence

<400> 27

ttggacaacg cccgtattgg tgggtacccc accctacccc ccatgttgcc ttttaag 57

<210> 28

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 28

ttggacaacg cccggtattg gtgggtcctg gacaccgact acta 44

<210> 29

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 29

ttggacaacg cccgtagtcc tggacaccga cacta 36

<210> 30

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 30

ttggacaacg cccgtatgtg ggtcctggac accgactact a 41

<210> 31

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 31

ttggacaacg cccgtatttg gtgggtcctg gacaccgact acta 44

Claims (10)

1. A sgRNA guide sequence of a specific targeting mouse G6pc gene is characterized in that the nucleotide sequence corresponding to the sgRNA is a sequence shown in SEQ ID NO. 2.

2. A method for editing a mouse G6pc gene using the sgRNA targeting sequence specifically targeting the mouse G6pc gene of claim 1, comprising the steps of:

step 1: synthesizing a forward oligonucleotide sequence by adding accg to the 5' end of the sgRNA guide sequence of the specific targeting mouse G6pc gene of claim 1;

meanwhile, according to the sgRNA guide sequence of the specific targeting mouse G6pc gene of claim 1, obtaining the corresponding DNA complementary strand, and adding aaac at the 5' end of the DNA complementary strand to synthesize a reverse oligonucleotide sequence;

annealing the forward oligonucleotide sequence and the reverse oligonucleotide sequence to form a double-stranded DNA fragment having a sticky end;

step 2: digesting a target vector pGL3-U6-sgRNA plasmid shown in SEQ ID NO.5 by using Bsa I restriction endonuclease to obtain a digestion product pGL3-U6-sgRNA-Bsa I;

and step 3: connecting the double-stranded DNA fragment with the sticky end obtained in the step 1 with the enzyme digestion product pGL3-U6-sgRNA-Bsa I obtained in the step 2, converting the connection product into competent escherichia coli, coating the competent escherichia coli on an ampicillin-resistant LB culture medium, culturing overnight at 37 ℃ for 20h, selecting a single clone, identifying a positive clone by sequencing with a universal primer U6 shown in SEQ ID No.6, shaking the positive clone, and extracting a plasmid to obtain pGL3-U6-G6pc-sgRNA plasmid;

and 4, step 4: co-transfecting mouse N2a cells with the pGL3-U6-G6pc-sgRNA plasmid obtained in the step 3 and the pST1374-NLS-flag-linker-Cas9 expression plasmid shown in SEQ ID NO.7, and obtaining positive sgRNA-Cas9 co-transfected cells after drug screening;

and 5: carrying out cell lysis on the positive sgRNA-Cas9 cotransfected cells obtained in the step (4), carrying out PCR amplification reaction on the DNA of the targeting site by taking the obtained cell lysis solution as a template, taking the PCR amplification product of the DNA of the targeting site to carry out Sanger sequencing, and if the targeting site has a set peak, primarily confirming that gene editing occurs;

step 6: TA cloning, sequencing and analyzing step 5 preliminarily confirms the genotype of the targeted site with gene editing and obtains mouse cells with the gene editing.

3. The method for editing mouse G6pc gene according to claim 2, wherein in step 1, the annealing reaction system is specifically: 10 μ M forward oligonucleotide, 5 μ L; 10 μ M reverse oligonucleotide, 5 μ L; 10 XT 7 EndonucleaaseI buffer, 2. mu.L; ddH₂O, 8 mu L; the reaction procedure is specifically as follows: 95 ℃ for 5 min; 95 ℃ to 85 ℃, and-1 ℃/cycle, for 10 cycles; 85 ℃ to 25 ℃ and-0.1600 cycles of annealing, and storing the annealing product at-20 ℃.

4. The method for editing mouse G6pc gene according to claim 2, wherein in step 2, the enzymatic digestion reaction system specifically comprises: pGL3-U6-sgRNA plasmid, 2. mu.g; 10 Xenzyme digestion buffer, 2 uL; bsa I restriction enzyme, 2. mu.L; supplemental ddH₂And O, setting the enzyme digestion reaction system at 37 ℃ for reaction for 3h until the total volume is 20 mu L.

5. The method for editing mouse G6pc gene according to claim 2, wherein the specific method for transforming competent Escherichia coli in step 3 is: adding 5 μ L of the ligation product into 30 μ L of competent Escherichia coli, mixing, standing on ice for 25min, performing water bath heat shock at 42 deg.C for 90s, cooling on ice for 2min, adding 150 μ L of LB liquid culture medium, rotating at 220 rpm, activating at 37 deg.C for 30min, and coating on the surface of LB solid culture medium with ampicillin resistance; in the LB culture medium containing ampicillin resistance, the concentration of ampicillin is 50 mug/mL; culturing overnight at 37 deg.C and 220 rpm; the extraction plasmid adopts a kit for extracting endotoxin-removing plasmid.

6. The method for editing mouse G6pc gene according to claim 2, wherein in step 4, the drugs are puromycin at a concentration of 50 μ G/ml and blasticidin at a concentration of 100 μ G/ml; the mouse N2a cells were inoculated and cultured in DMEM complete medium containing 10% v/v fetal calf serum at 37 ℃ and 5% CO before transfection₂Culturing in an incubator, replacing fresh culture medium every 2d-3d, digesting with 0.25% trypsin after the cell confluence reaches 80-90%, then passing through a 6-well plate, and transfecting after 18-20 h and when the cell confluence reaches 60-70%.

7. The method for editing mouse G6pc gene according to claim 2, wherein in step 5, the PCR amplification reaction system of the target site DNA comprises: 2 mu L of cell lysate, 1 mu L of upstream primer, 1 mu L of downstream primer, 2 mu L of dNTP mix, 1 mu L of TaKaRa Ex Taq, 2.5 mu L of 10 XEx Taq Buffer and 25 mu L of sterilized water; the PCR amplification reaction of the target site DNA is carried out by the following procedures: 95 ℃ for 5 min; 95 ℃, 20s, 72 ℃, 20s, -1 ℃/cycle, 72 ℃, 25s, for 10 cycles; 25 cycles of 95 ℃, 20s, 62 ℃, 20s, 72 ℃, 25 s; 72 ℃, 5min, 16 ℃, infinity.

8. The method for editing mouse G6pc gene according to claim 7, wherein the sequence of the upstream primer is shown in SEQ ID No. 8; the sequence of the downstream primer is shown as SEQ ID NO. 9.

9. The method for editing mouse G6pc gene according to claim 2, wherein in step 6, the TA clone sequencing specifically comprises: performing gel recovery and purification on the PCR amplification product of the targeted site obtained in the step 5, connecting the purified DNA, and then performing metal bath at 16 ℃ for 1h to obtain a connection product; and transforming the ligation product into competent escherichia coli, coating the competent escherichia coli on an ampicillin-resistant LB solid culture medium, performing overnight culture at 37 ℃ for 20h, performing colony PCR reaction verification, screening out positive clones, and performing Sanger sequencing on the positive clones.

10. The method for editing mouse G6pc gene according to claim 9, wherein the linked reaction system is: PCR purified product 40ng, Solution I2.5. mu.L and PMD19 carrier 0.5. mu.L, and water is added to make up to 5. mu.L; the specific method for transforming the competent escherichia coli comprises the following steps: adding 5 μ L of the ligation product into 30 μ L of competent Escherichia coli, mixing, standing on ice for 25min, performing water bath heat shock at 42 deg.C for 90s, cooling on ice for 2min, adding 150 μ L of LB liquid culture medium at 220 rpm and 37 deg.C for 30min, and coating on the surface of LB liquid culture medium containing ampicillin resistance; in the LB culture medium containing ampicillin resistance, the concentration of ampicillin is 50 mug/mL; the colony PCR reaction system comprises: 1 mu L of colony aqueous solution, 5 mu L of Premix Taq enzyme, 0.5 mu L of upstream primer, 0.5 mu L of downstream primer and 3 mu L of sterilized water; the procedure of the colony PCR reaction was: 95 ℃ for 5 min; 95 ℃, 20s, 60 ℃, 20s, 72 ℃, 25s, 26 cycles; 72 ℃ for 5 min; the sequence of the upstream primer is shown as SEQ ID NO. 10; the sequence of the downstream primer is shown as SEQ ID NO. 11.

Images