CN116790604B - sgRNA and CRISPR/Cas9 vector as well as construction method and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of molecules, and particularly provides a sgRNA and CRISPR/Cas9 vector as well as a construction method and application thereof, wherein the sgRNA comprises APOE-sgRNA2 with a nucleotide sequence shown as SEQ ID NO.2 or SEQ ID NO.10 and LDLR-sgRNA3 with a nucleotide sequence shown as SEQ ID NO.7 or SEQ ID NO. 15. According to the CRISPR/Cas9 system constructed by specific sgRNA, the editing efficiency of the double alleles of the APOE and the LDLR is more than 60%, the editing efficiency of the double alleles of the APOE and the LDLR is more than 30%, the editing efficiency of the double alleles of the APOE and the LDLR is improved, the construction cost of the double alleles of the APOE and the LDLR knocked out cell strain is reduced, and the practical popularization and application values are specific.
Description
Technical Field
The invention belongs to the technical field of molecules, and particularly relates to an sgRNA and CRISPR/Cas9 vector, a construction method thereof and application thereof in APOE and LDLR double-allele knockout cell strains.
Background
Hyperlipidemia refers to a condition of high blood lipid level, which can directly cause some diseases seriously harming human health, such as atherosclerosis, coronary heart disease, pancreatitis, etc. Low density lipoprotein (Low Density Lipoprotein Receptor, LDLR), apolipoprotein E (ApolipoproteinE, apoE) is currently the major gene for hyperlipidemia. In recent years, by editing APOE and LDLR genes, a hyperlipidemia animal model is constructed, and the model is used for researching hyperlipidemia to continuously develop greatly. For example, APOE edited bama miniature pigs are prepared from university of south Beijing medical science, APOE and LDLR edited bama miniature pigs are prepared from institute of animal sciences of the national academy of agricultural science, PCSK9 gene edited wuzhishan miniature pigs are prepared from institute of Beijing livestock veterinarian of the national academy of agricultural science, and after weaning, spontaneous plasma cholesterol (TC) levels are 3 to 5 times that of wild type, and low density lipoprotein cholesterol (LDL-C) is 2 to 3 times that of wild type.
Gene editing relies on genetically engineered CRISPR/Cas9, also known as "molecular scissors",
the gene can be used for eliminating, adding, activating or inhibiting target genes of other organisms, and provides a wide prospect for treating human diseases. Until the complete realization of the disease research landscape based on gene editing, the challenges facing researchers have also long existed. During the last decades, scientists have devised a variety of gene delivery technologies in studying how to deliver proteins and nucleic acids, playing a vital role in the process of gene editing, including adeno-associated viral and lentiviral vectors for gene therapy, as well as lipid nanoparticles and non-viral vectors for delivering proteins and nucleic acids. However, these vectors have low delivery efficiency, insufficient ability to target specific tissues, and low gene editing efficiency. For example CN113046388A discloses a CRISPR/Cas9 system for pig APOE and LDLR gene editing, which is only 61% efficient for APOE editing and only 33% efficient for LDLR gene editing. The efficiency of gene editing is low, resulting in a hyperlipidemia animal model that is time consuming, laborious and costly.
Disclosure of Invention
In order to solve the problems, the invention provides an sgRNA which comprises APOE-sgRNA2 with a nucleotide sequence shown as SEQ ID NO.2 or SEQ ID NO.10 and LDLR-sgRNA3 with a nucleotide sequence shown as SEQ ID NO.7 or SEQ ID NO. 15.
The invention also provides a CRISPR/Cas9 vector, which is a vector connected with double-stranded DNA corresponding to the APOE-sgRNA2 and a vector connected with double-stranded DNA corresponding to the LDLR-sgRNA3;
the nucleotide sequence of the APOE-sgRNA2 is shown as SEQ ID NO.2 or SEQ ID NO. 10; the nucleotide sequence of the LDLR-sgRNA3 is shown as SEQ ID NO.7 or SEQ ID NO. 15.
Further, the vector is a pX458 plasmid vector.
The carrier connected with double-stranded DNA corresponding to the APOE-sgRNA2 can be expressed in cells to generate the APOE-sgRNA2; the vector connected with double-stranded DNA corresponding to the LDLR-sgRNA3 can be expressed in cells to generate the LDLR-sgRNA3;
wherein, APOE-sgRNA2 is a sgRNA guiding Cas9 to edit APOE genes at fixed points;
LDLR-sgRNA3 is a sgRNA that directs Cas9 to site-directed editing of LDLR genes.
The invention also provides a construction method of the CRISPR/Cas9 carrier, which comprises the following steps:
taking double-stranded DNA corresponding to the APOE-sgRNA2, and inserting the double-stranded DNA into a pX458 plasmid vector; taking double-stranded DNA corresponding to LDLR-sgRNA3, and inserting the double-stranded DNA into a pX458 plasmid vector to obtain the double-stranded DNA;
the nucleotide sequence of the APOE-sgRNA2 is shown as SEQ ID NO.2 or SEQ ID NO. 10; the nucleotide sequence of the LDLR-sgRNA3 is shown as SEQ ID NO.7 or SEQ ID NO. 15.
The invention also provides application of the CRISPR/Cas9 vector in preparing APOE and LDLR double-allele knockout cell strains or animals.
Further, the cell beads comprise wuzhishan pig ear fibroblasts; the animals include Wuzhishan pigs.
The invention also provides a construction method of the APOE and LDLR double-allele knockout cell strain, which comprises the following steps:
and (3) cotransfecting the wuzhishan pig ear fibroblasts with the CRISPR/Cas9 vector, enriching and culturing the cells with green fluorescence, and obtaining the APOE and LDLR double-allele knocked-out cell strain by sequencing and identification.
The invention has the beneficial effects that:
the CRISPR/Cas9 system constructed by specific sgRNA has the efficiency of editing the double alleles of APOE or LDLR more than 60%, the efficiency of editing the double alleles of APOE and LDLR more than 30%, the efficiency of editing the double alleles of APOE and LDLR is improved, the construction cost of the double allele knockout cell strain of APOE and LDLR is reduced, and the practical popularization and application values are specific.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 APOE-sgRNA editing efficiency validation sequencing results;
FIG. 2 LDLR-sgRNA editing efficiency validation sequencing results;
FIG. 35 single cell clone sequencing results of two-allele editing of APOE and LDLR genes;
FIG. 4 APOE and LDLR gene editing clone Wuzhishan pig ear fibroblasts, on day 7, were spiked;
FIG. 5 APOE and LDLR gene editing clone five-finger mountain pig ear fibroblast postpass cell growth conditions;
FIG. 6 development of APOE and LDLR gene editing cloned wuzhishan pig ear fibroblasts.
Detailed Description
Example 1 ApoE, LDLR Dual Gene editing cell lines and application Studies
1.1 design of CRISPR/Cas9 knockout vector targeting APOE and LDLR genes and verification of editing efficiency thereof
With reference to the Gene sequence information of porcine APOE (Gene ID: 396801) and LDLR (Gene ID: 397576) in NCBI, a website (http:// crispr. Mit. Edu /) was designed on line using single-stranded guide RNA (sgRNA) to design 4 sgRNA targeting sites for the APOE second exon and LDLR third exon, respectively, two (coding strand and non-coding strand) short DNA sequences corresponding to the sgRNA were synthesized and cohesive ends were added, annealed to double strands (94 ℃, 10 min; 37 ℃, 10 min,4 ℃), and stored. And (3) carrying out enzyme digestion and recovery on the pX458 vector (with GFP fluorescent marker) by using Bbs I restriction endonuclease, connecting the linearized vector after enzyme digestion and recovery with a DNA sequence which is annealed and paired into double chains, further converting and plating, and obtaining the CRISPR/Cas9 vector with accurate connection after sequencing confirmation (shown in the following table 1).
The well-grown wuzhishan pig Ear Fibroblasts (EF) were digested with 0.1% pancreatin, centrifuged at 1200rpm, and the supernatant was discarded. Cells were resuspended with 100. Mu.l of electrotransfer fluid every 5X 10 5 Individual cells were mixed with a single CRISPR/Cas9 vector, transferred to an electrocuvette after mixing, electrotransfected with a Lonza nuclear transfecter, and the shocked cells were inoculated into a 100mm cell culture dish and cultured with DMEM containing 20% FBS for a further 48h. The electrotransformed cells were digested into single cell suspensions by 0.1% pancreatin digestion, and flow sorted to collect GFP-bearing green fluorescent cells, respectively. Genomic DNA of GFP positive cells was extracted and PCR amplified. Primers were designed on both sides of the two sgRNA sites of action, as follows:
SUS-APOE-ID-F（SEQ ID NO .17）：5’- TTCTAATGCGTTGTTGCCTGG-3’；
SUS-APOE-ID-R（SEQ ID NO .18）：5’- ACAAGGACAGAAGGAAACCCG-3’；
the size of the product is 556bp;
SUS-LDLR-ID-F（SEQ ID NO .19）:5’- AAAGGGGAGCTCGCTACTCA-3’；SUS-LDLR-ID-R（SEQ ID NO .20）：5’- GAGCCTCACTTCATGAAAGACAT-3’；
the size of the product is 699bp;
the PCR amplification system was as follows: 1. Mu.L of each of the upstream and downstream primers (10 pmoL/. Mu.L); genomic DNA 0.5. Mu.g, premix LA Taq 10. Mu.L, sterilized distilled water was added to 20. Mu.L. PCR reaction conditions: 95. 5min at the temperature; (95 ℃ C. 30 s, 53 ℃ C. 30 s, 68 ℃ C. 20 s) x 32cycles; 68. 5min at the temperature; 16. preserving at the temperature. And (3) purifying and recovering the PCR product, performing TA cloning, culturing for 12 hours, randomly sequencing more than 25 single colonies, comparing the sequencing result with a wild type gene sequence, and counting the editing efficiency of CRISPR/Cas9 vectors of the sgRNAs. The results are shown in Table 1 below, and the sequencing results are shown in FIGS. 1 and 2.
TABLE 1 sgRNA of APOE and LDLR genes and editing efficiency thereof
Note that: SEQ ID NO. 1-8 are nucleotide sequences of sgRNA on a vector, and SEQ ID NO. 9-16 are nucleotide sequences of sgRNA in cells.
1.2 cell transfection Screen and monoclonal identification
And F, co-transfecting the APOE-sgRNA2 and the LDLR-sgRNA3 vector (with GFP fluorescent markers) with highest effective editing efficiency, enriching green fluorescent cells by a flow cytometer, inoculating 100 cells/dish into a 100mm cell culture dish, culturing for about 15 days, transferring single cell clones into a 48-hole cell culture plate for continuous culture, and taking part of cells for gene knockout identification when the cells grow to 80% -90%. Extracting cell genome DNA by a cell lysate method, analyzing the gene knockout type by PCR sequencing, and freezing and storing single cell clones of which the APOE and LDLR double alleles are knocked out for subsequent somatic cell nuclear transfer experiments. 25 cloning points were identified, the efficiency of editing the APOE and LDLR double alleles and the efficiency of editing the double alleles are shown in Table 2 below, the efficiency of editing the double alleles of the APOE and LDLR is 20%, and 5 single cell clones of editing the double alleles of the APOE and LDLR are obtained, and genotypes of the single cell clones are shown in Table 3. The sequencing results are shown in FIG. 3.
TABLE 2 APOE and LDLR double allele editing efficiency and two-gene double allele editing efficiency
TABLE 3 genotypes of single cell clones with APOE and LDLR double allele knockouts
1.3 preparation and identification of cloned pig edited by APOE and LDLR genes
Randomly selecting 3APOE and LDLR double-gene editing type cells (namely A19, A3 and A4) as nuclear donors, taking the in-vitro mature oocytes as acceptors after enucleation, and carrying out somatic cell nuclear transfer to obtain reconstructed embryos. After the reconstructed embryo is cultured for 12-24 hours, the embryo with good development state is transferred into the fallopian tube of a backup sow with natural oestrus for 0-1d by an operation method, the gestation condition is detected by the embryo transfer 28d and 60d B, and the recipient sow is born about 114d gestation. Collecting piglet ear tissue samples, extracting genome DNA, sending PCR amplified products to sequence, and identifying and analyzing the types of piglet APOE and LDLR gene editing.
The embryo transfer and pregnancy delivery conditions of the 3APOE and LDLR double-gene editing type cells are shown in table 4, the average embryo transfer numbers of each generation of pregnant sows of a19, A3 and A4 cells are 91,83 and 117 respectively, and the pregnancy rate is more than 60%. In addition, the numbers of piglets produced by the a19, A3 and A4 cell clones, their corresponding numbers and genotypes are shown in table 5, and the genotypes and cells of the piglets are consistent.
Table 4 3 embryo transfer and gestation delivery conditions of APOE and LDLR double-gene editing cells
TABLE 5 numbering and genotype of APOE and LDLR Gene-edited cloned piglets
1.4 Phenotype detection of APOE and LDLR gene editing clone wuzhishan pigs:
APOE and LDLR gene-edited and Wild (WT) wuzhishan pig blood of 2 and 5 months of age, respectively, were collected, centrifuged at 1500 rpm for 15min and serum was collected and tested for blood lipid index, i.e., total Cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C). Blood lipid test results are shown in the following tables 6 and 7, and the serum TC and LDL-C levels of piglets obtained by A3, A4 and A19 cell clones are significantly higher than those of WT pigs at 2 months and 5 months of age, and the TG and HDL-C of piglets are not significantly different from those of WT pigs, so that the APOE and LDLR gene editing type pigs obtained by A3, A4 and A19 cell clones are proved to have stable hyperlipidemia phenotype without high-fat diet interference. Specifically, the APOE and LDLR gene editing type pigs obtained by cloning A19 cells have TC 8-9 times that of WT pigs and LDL-C11-13 times that of wild type pigs.
Table 6 2 month APOE and LDLR Gene editing and wild type wuzhishan pig blood lipid index detection results
Table 75 month APOE and LDLR Gene editing and wild type wuzhishan pig blood lipid index detection results
1.5 Primary isolated culture of APOE and LDLR gene editing clone wuzhishan pig ear fibroblast
Wiping pig ears with alcohol cotton, roughly cutting off skin hair, shearing off 1-2cm of ear tissue with large scissors, and sterilizing with iodine wine at pig ear wound; fine shearing and scraping off hair on the surface of the ear tissue by a blade, soaking in alcohol for 2-5min, transferring to DMEM, and carrying back to a laboratory at low temperature. And (5) placing the ear tissue into 75% alcohol, soaking and cleaning for 2-3 min. The alcohol burner burns the blade and the ophthalmic forceps, further scrapes the ear hairs in the culture dish, moves the treated ear tissue into a new culture dish, cuts the tissue from it, exposes cartilage, fat, etc. in the tissue, and scrapes off only the epidermis. The epidermis was repeatedly immersed in 5% diabody DMEM (placed in several dishes respectively), the ophthalmologic forceps tapped the epidermis, and the impurities were shaken until the liquid was clear. The epidermis is transferred into an empty dish, a few drops of DMEM are dripped into the dish, the epidermis is kept moist, and the epidermis is sheared by ophthalmic scissors until the epidermis is less than or equal to 1mm. Adding proper amount of fetal bovine serum into the sheared epidermis, uniformly mixing, equally dividing the tissue fragments on one surface of a T-25 bottle (marked with cell types and dates) with an inclined surface, uniformly spreading the tissue fragments, turning over, adding 5mL of DMEM (medium solution) containing 20% FBS and 5% PS along the other surface of the T-25 bottle, enabling one surface of the tissue block to be upward, putting the tissue block into an incubator, turning over the bottle for about 8 hours, namely gently turning the T-25 bottle upside down, and enabling the culture solution to soak the tissue. The whole culture solution is changed once every other day, and more cells generally climb out (the cell climbing out is shown in fig. 4) 7 days after bottle turning, and when a large amount of cells grow out, the cells are passaged, used or frozen.
(1) Subculture of APOE and LDLR gene editing clone wuzhishan pig ear fibroblasts
When the primary cells in the step (1) grow out in a large quantity, pouring out the primary liquid culture medium, washing 1 time by using DPBS (Du's phosphate buffer solution) added with 1% green chain double antibody and preheated to 37.5 ℃, adding 0.1% trypsin to digest the cells, adding 2 times of complete culture medium (DMEM containing 20% FBS), stopping digestion, sucking into single cell suspension, and beating according to the ratio of 5 to 10 6 Culturing in 10cm cell culture dish at 37.5deg.C under saturated humidity with 5% CO 2 In the incubator of (a), APOE and LDLR genes edit and clone the growth condition of cells after transmission of the five-finger pig ear fibroblasts, as shown in figure 5, the cells are in a typical long fusiform, triangle and the like.
(2) Freezing, recovering and survival rate determination of APOE and LDLR gene editing clone wuzhishan pig ear fibroblasts
When the confluence of the passage cells is nearly 90%, the waste culture medium is removed, DPBS is washed, 0.1% pancreatin is digested into single cell suspension, the single cell suspension is transferred into a freshly sterilized 15ml centrifuge tube, the cells are gathered at the bottom of the tube in a table centrifuge at 1200rpm for 5min, the supernatant is thoroughly removed, and the cell freezing solution is added. The formula of the frozen stock solution is DMEM: fetal bovine serum: dmso=6:3:1, after gently mixing the cells with the frozen stock solution, transferring into a freezing tube, noting the date and sample name, freezing the freezing tube, freezing the frozen stock, and transferring the cells into liquid nitrogen for long-term storage the next day according to the steps of 4 ℃,30 min- & gtminus 20 ℃,30 min- & gtminus 80 ℃ in an ultralow temperature refrigerator overnight.
And taking out the frozen cells of each male and female tube 1 from the liquid nitrogen, and rapidly placing the frozen cells in a warm water bath at 37.5 ℃ to continuously shake the frozen cells for melting. Sucking out cell suspension, adding into 15ml sterile centrifuge tube, adding 5 times volume of complete culture medium (DMEM containing 20% FBS), mixing by gentle shaking, collecting cells at the bottom of tube at 1200rpm for 5min on table centrifuge, thoroughly removing supernatant, adding appropriate volume of complete culture medium (DMEM containing 20% FBS), mixing by gentle shaking, sucking out cells, adding into culture flask, culturing at 37.5deg.C, saturated humidity, and 5% CO 2 Is arranged in the incubator. Cell viability was calculated using trypan blue exclusion staining. Taking out a proper amount of cells, adding the cells into trypan blue dye solution, and counting the number of blue cells under an optical microscope, wherein the calculation formula of the cell viability is as follows: survival = (1-blue cell number/total cell number) ×100%, cell survival is: 89.2%.
(3) Reprogramming ability of APOE and LDLR gene editing clone wuzhishan pig ear fibroblasts
And (3) the cells separated in the step (1) are injected into the enucleated in-vitro mature pig oocyte by a nuclear transfer method, and after fusion, activation and in-vitro culture, the cleavage and blastula development conditions of the reconstructed embryo are observed under a microscope, and the development capacity, namely the reprogramming capacity, of the reconstructed embryo is evaluated. The development of APOE and LDLR gene editing clone wuzhishan pig ear fibroblasts is shown in FIG. 6 below, with a cleavage rate of 77.6% and a blastula rate of 23.7%.
In conclusion, the CRISPR/Cas9 system constructed by specific sgRNA has the double allele editing efficiency of more than 60% in both APOE and LDLR, the double allele editing efficiency of more than 30% in both APOE and LDLR, the success rate of constructing animal models of double allele knockout cell strains of APOE and LDLR is improved, the construction cost of the double allele knockout cell strains of APOE and LDLR is reduced, and the practical popularization and application values are specific.
Claims (7)
1. A set of sgrnas, characterized in that: it comprises APOE-sgRNA2 with a nucleotide sequence shown as SEQ ID NO.10 and LDLR-sgRNA3 with a nucleotide sequence shown as SEQ ID NO. 15.
2. A set of CRISPR/Cas9 vectors characterized by: it is a vector to which double-stranded DNA corresponding to APOE-sgRNA2 is ligated, and a vector to which double-stranded DNA corresponding to LDLR-sgRNA3 is ligated;
the nucleotide sequence of the APOE-sgRNA2 is shown as SEQ ID NO. 10; the nucleotide sequence of the LDLR-sgRNA3 is shown as SEQ ID NO. 15.
3. The CRISPR/Cas9 vector according to claim 2, characterized in that: the vector is a pX458 plasmid vector.
4. A method of constructing the CRISPR/Cas9 vector of claim 2 or 3, comprising the steps of:
taking double-stranded DNA corresponding to the APOE-sgRNA2, and inserting the double-stranded DNA into a pX458 plasmid vector; taking double-stranded DNA corresponding to LDLR-sgRNA3, and inserting the double-stranded DNA into a pX458 plasmid vector to obtain the double-stranded DNA;
the nucleotide sequence of the APOE-sgRNA2 is shown as SEQ ID NO. 10; the nucleotide sequence of the LDLR-sgRNA3 is shown as SEQ ID NO. 15.
5. Use of the CRISPR/Cas9 vector of claim 2 or 3 in the preparation of an APOE and LDLR double allele knock-out cell line.
6. Use according to claim 5, characterized in that: the cell strain comprises five-finger mountain pig ear fibroblasts.
7. A method for constructing a cell line from which an APOE and LDLR double allele is knocked out, comprising the steps of: it comprises the following steps:
the CRISPR/Cas9 vector of claim 2 is used for transfecting wuzhishan pig ear fibroblasts, green fluorescent cells are enriched and cultured, and the cell strain with APOE and LDLR double alleles knocked out is obtained through sequencing and identification.
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