CN116694683A - CRISPR-Cas9 system for targeting downstream sequence of pig COL1A1 gene and application thereof - Google Patents
CRISPR-Cas9 system for targeting downstream sequence of pig COL1A1 gene and application thereof Download PDFInfo
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Abstract
The invention provides a CRISPR-Cas9 system for targeting a downstream sequence of a pig COL1A1 gene and application thereof. The system comprises a gRNA that specifically recognizes the 3' flanking region of the porcine COL1A1 gene and a donor vector for integration of the exogenous sequence into the site. The gRNA provided by the invention can recognize that 3' flanking regions of a pig COL1A1 gene are shown as SEQ ID NO:1, and a target sequence shown in 1. Double-strand break can be generated at a specific position of the region through the mediation of the gRNA, the cutting efficiency can reach 38.2%, and the site-directed integration of the exogenous sequence at the position can be realized through simultaneous transfer into the Donor-1, donor-2 or Donor-3 vector provided by the invention.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a CRISPR-Cas9 system for targeting a downstream sequence of a pig COL1A1 gene and application thereof.
Background
Transgenic technology has important value in the fields of gene function research, biomedical research and agriculture, but conventional transgenic technology is accompanied by random integration problems, and causes a number of adverse effects such as gene silencing and the like. Researchers have been struggling to find ways to solve these problems, one of which is to integrate exogenous gene sequences into the genome at a site-specific site that mediates the transcriptional activation of stable, efficient expression of exogenous genes, i.e., a safe site in the genome.
Pigs are important economic animals, not only in the agricultural livestock industry, but also in the medical model animals. However, the number of the identified genome safety sites in the genome of the pig is small, and the development requirements of pig breeding and a transgenic pig model for medical use are difficult to meet.
Disclosure of Invention
The invention aims to provide a CRISPR-Cas9 system for targeting a downstream sequence of a pig COL1A1 gene and application thereof.
In order to achieve the aim of the invention, in a first aspect, the invention provides a pig COL1A1 gene downstream sequence targeting vector based on a CRISPR-Cas9 system, wherein the targeting vector is a pX330 vector containing gRNA.
The DNA sequence of the gRNA recognition site is as follows: 5'-ACGACCCTCCAGTTCGCCTAGGG-3' (SEQ ID NO: 2) flanking the 3' region of the porcine COL1A1 gene. The accession number of the pig COL1A1 gene in NCBI is 100738123.
The gRNA is a guide RNA, is an important component part in a CRISPR gene knockout knock-in system, is combined with Cas9 protein, and guides Cas9 enzyme to target genomic DNA for cutting. The gRNA for specifically recognizing the 3' -flanking region of the pig COL1A1 gene provided by the invention has a nucleotide sequence 5'-ACGACCCUCCAGUUCGCCUA-3' (SEQ ID NO: 8) responsible for recognizing a target fragment region. Through the mediation of the gRNA, double-strand break can be generated in a 3' -flanking region of the COL1A1 gene on a Cas9 targeted pig genome, the cutting efficiency can reach 38.2%, editing of the site, such as base replacement, or insertion of an exogenous gene expression frame in the site is realized, and stable and efficient expression of the exogenous gene is realized.
In a second aspect, the invention provides a donor vector I, which comprises a left homology arm of a targeting region of a downstream sequence of a pig COL1A1 gene, an exogenous gene expression frame and a right homology arm of the targeting region of the downstream sequence of the pig COL1A1 gene which are connected in sequence; or alternatively, the process may be performed,
comprises a left homology arm of a downstream sequence targeting region of the pig COL1A1 gene, an insulator, an exogenous gene expression frame, an insulator and a right homology arm of the downstream sequence targeting region of the pig COL1A1 gene which are connected in sequence.
In the present invention, the exogenous gene expression cassette comprises a promoter, an exogenous gene sequence and a terminator.
Preferably, the nucleotide sequences of the left and right homology arms of the downstream sequence targeting region of the pig COL1A1 gene are respectively shown in SEQ ID NO: 6. shown at 7.
In a third aspect, the invention provides a donor vector II comprising the above gRNA and an exogenous gene expression cassette.
In a fourth aspect, the invention provides a donor vector III comprising a gRNA-foreign gene expression cassette-gRNA linked in sequence.
The aforementioned donor vector, the backbone vector may be pUC57.
In a fifth aspect, the invention provides a CRISPR-Cas9 system targeting the 3' flanking region of the pig COL1A1 gene, comprising the targeting vector and the donor vector (I, II or III).
In a sixth aspect, the invention provides the use of the CRISPR-Cas9 system in swine gene editing.
In a seventh aspect, the invention provides a method for targeting the 3' -flanking region of the pig COL1A1 gene for site-directed integration of exogenous genes, wherein the targeting vector and the donor vector (I, II or III) are transfected together to obtain pig embryo fibroblasts, and positive clones are obtained by screening.
After transfection of pig embryo fibroblasts and screening to obtain site-directed integration clones, the pig with site-directed integration exogenous genes is obtained through somatic cloning technology and embryo transfer technology.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
the gRNA for specifically recognizing the downstream sequence of the pig COL1A1 gene provided by the invention can recognize that the 3' flanking region of the pig COL1A1 gene is shown as SEQ ID NO:1, and a target sequence shown in 1. Through the mediation of the gRNA, cas9 can target a specific position of a 3' flanking region of the COL1A1 gene on a No. 12 chromosome of a pig genome and generate double-strand break, and the cutting efficiency can reach 38.2%. Meanwhile, the exogenous gene sequence can be integrated into the site in a fixed point mode by matching with the Donor-1, donor-2 or Donor-3 vector in the system, so that stable and efficient expression of the exogenous gene sequence in various tissues of pigs is realized.
Drawings
FIG. 1 is a graph showing the direct sequencing peaks of the gRNA efficiency detection PCR product of example 1 of the present invention.
FIG. 2 is a graph showing the cleavage of the gRNA efficiency assay T7E1 in example 1 of the present invention.
FIG. 3 is a schematic diagram showing site-directed integration of exogenous genes using gRNA, cas9 protein and Donor-1 vector carrying exogenous genes in example 2 of the present invention.
FIG. 4 shows the flow-sorting of PEF cells transfected with gRNA, cas9 protein and Donor-1 vector carrying exogenous gene in example 2 of the present invention, and the proportion of GFP positive cells was examined.
FIG. 5 is a schematic representation of the positions of PCR primers for monoclonal cell identification of flow-sorted GFP-positive PEF cells in example 2 of the present invention.
FIG. 6 is a partial result of monoclonal cell identification of GFP-positive PEF cells flow-sorted in example 2 of the present invention, with lanes M representing DNA markers; lanes 1-22 represent monoclonal PEF cells; WT lane represents wild-type PEF cells; the water lane represents a negative control without template; a, amplifying GFP fragments, wherein the size of a target band is 135bp; b, left Junction PCR, band size is 1240bp; c, right Junction PCR, band size 1290bp.
FIG. 7 is a schematic diagram showing the integration of the Donor-2 vector in example 3 of the present invention, wherein GOI represents the expression cassette of the foreign gene.
FIG. 8 is a schematic diagram showing the integration of the Donor-3 vector in example 3 of the present invention, wherein GOI represents the expression cassette of the foreign gene.
FIG. 9 is a schematic diagram showing the identification of binding PCR after Donor-2 vector integration in example 3 of the present invention, wherein a represents forward integration and b represents reverse integration.
FIG. 10 is a schematic diagram showing the identification of binding PCR after Donor-3 vector integration in example 3 of the present invention, wherein a represents forward integration and b represents reverse integration.
FIG. 11 shows the structure of the Donor-1 vector in example 2 of the present invention.
FIG. 12 shows the structure of the Donor-2 vector in the preferred embodiment 3 of the present invention.
FIG. 13 shows the structure of the Donor-3 vector in the preferred embodiment 3 of the present invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
Porcine fetal fibroblasts (porcine embryonic fibroblast, PEF) tested in the following examples were prepared as follows: removing heads, tails, limbs, viscera and bones of fetuses from 35-37 day old fetuses of large white pigs, and cleaning blood. Continuously shearing the fetus for 30min by using elbow ophthalmic scissors to ensure sufficient shearing, sucking the sheared fetal tissue into a 15mL centrifuge tube by using a blue gun head of the shearing head, adding 5mL of complete culture medium, naturally settling for several minutes, removing the upper solution, adding a few drops of FBS into a lower tissue block, sucking out by using a 15cm glass Pasteur pipe bent at a position of 1cm at the tip, spreading in two T75 culture flasks, placing the bottoms of the two culture flasks upwards, adding 15mL of complete culture solution at the opposite side, carefully overturning the culture flasks after 6-8 h, immersing the tissue block into the culture solution, changing the solution once every two days, and freezing for later use after the T75 culture flasks are full of cells.
EXAMPLE 1 construction of Cas9/gRNA expression vector specifically recognizing target site of 3' flanking region of COL1A1 Gene and gRNA Activity detection
1. Construction of Cas9/gRNA expression vectors
Firstly, selecting a section of sequence of a target site of a 3' -flanking region of a pig COL1A1 gene as a target sequence, wherein the target sequence is shown as SEQ ID NO: 1.
Designing sequences responsible for identifying target fragment regions in gRNAs according to target sequences, scoring the sequences according to experience of the research team, selecting three gRNAs responsible for identifying target fragment regions with highest scores, and optimizing according to 5' -end sequences, wherein the sequences of the gRNAs are named as F-sg1, F-sg2 and F-sg3, and the sequences of the F-sg1 are shown as SEQ ID NO:3, the F-sg2 sequence is shown as SEQ ID NO:4, the F-sg3 sequence is shown as SEQ ID NO: shown at 5.
F-sg1：5′-ACGACCCTCCAGTTCGCCTA-3′(SEQ ID NO：3)；
F-sg2：5′-AGTTCTCTCCCTAGGCGAAC-3′(SEQ ID NO：4)；
F-sg3：5′-AGGCGAACTGGAGGGTCGTC-3′(SEQ ID NO：5)。
2. Construction of gRNA targeting vector
According to the three gRNA sequences, the three gRNA sequences are connected into a pX330 vector after BbsI digestion to obtain a pX330-gRNA vector.
3. 5 mug of the constructed pX330-gRNA vector is electrically transfected into PK15 cells, cell DNA is extracted after 48 hours of transfection, PCR reaction is carried out by using the following primers, and a target region of a 3' -flanking region of a pig COL1A1 gene is amplified. The PCR primer sequences were as follows (amplification product length 562 bp):
F-562：5′-CGATGCCACCAACTCTCTC-3′；
R-562：5′-CAGAGGGTGAGCGAAAAGG-3′。
4. sequencing of PCR products to detect gRNA Activity
And carrying out first-generation sequencing on the PCR amplification product, and detecting whether a hybrid peak appears at the target site of the gRNA, wherein the appearance of the hybrid peak indicates that the gRNA has the activity of cutting double-stranded DNA of the target site. As shown in FIG. 1, the sequencing peak diagram of the targeting site of the F-sg1 sample shows obvious impurity peaks, and neither F-sg2 nor F-sg3 shows impurity peaks.
5. Detection of gRNA Activity by T7E1 cleavage method
And (3) after the PCR amplification product is denatured and annealed, adding T7E1 enzyme and buffer solution to carry out enzyme digestion reaction, carrying out polyacrylamide gel electrophoresis on the enzyme digestion product, and calculating the cutting efficiency through imageJ software. As shown in FIG. 2, after T7E1 cleavage, the cut-out band appeared in the PCR product of gRNA-1, indicating that F-sg1 generated sequence mutation in the targeting region, and the band brightness was calculated by ImageJ software to give F-sg1 cleavage efficiency of about 38.2%. No mutation was detected in F-sg2 and F-sg 3.
From the above experiments, it can be seen that F-sg1 mediated the knockout of Cas9 protein is best.
Example 2 site-directed integration of exogenous genes by Donor-1
The structure of the Donor-1 vector is shown in FIG. 3, and the vector pUC57 is taken as a skeleton, and a left homology arm sequence (LA) and an exogenous gene expression frame (the expression frame generally comprises a promoter, a coding sequence, a transcription termination signal and other elements) are sequentially inserted into a multi-cloning site.
The nucleotide sequences of the left and right homologous arms of the pig COL1A1 gene are respectively shown in SEQ ID NO: 6. shown at 7.
The primary porcine embryo fibroblasts were resuscitated into 6cm dishes the day before transfection, and cell transfection was performed when the cells reached 70% -80% confluence. The cell transfection method was based on nuclear transfer instrument electrotransfection using Basic Primary Fibroblasts Nucleofector (Lonza) electrotransfection under Amaxa Nucleofector (Lonza) single well nuclear transfection system.
The specific operation flow is as follows:
a. collecting cells, and adjusting the number of cells to 1×10 6 Tube, 200g centrifuge for 5min, and remove the culture solution as clean as possible.
b. 100. Mu.L of the electrotransfection reagent was added to resuspend the cells, and 5. Mu.g of the Cas9/F-sg1 expression vector plasmid and 5. Mu.g of the linearized Donor-1 plasmid (containing the 3' -flanking region homology arm sequence of the COL1A1 gene and GFP expression cassette) were added, and the electrotransfection system (including cells, transfection reagent, plasmid) was slowly added to the electrotransfection cup along the wall to avoid air bubbles and reduce transfection efficiency. The optimal transfection procedure for PEF cells was U-023, where the viability and transfection efficiency of cells were higher than for the other procedures.
c. After transfection was completed, 500. Mu.L of complete medium (20% FBS+DMEM) was added to the electrotechnical flask, the cells were gently blown off and then transferred to a pre-warmed 6cm dish containing 5mL of culture medium at 37℃with 5% CO 2 Culturing in an incubator. After 72h of electrotransfection, the cells of the blank control group (not transfected) and the cells of the experimental group were aspirated off the medium, washed twice with PB S, and digested with an appropriate amount of trypsinAnd (5) performing chemical treatment for 2min. The digested cells were transferred to a 15mL centrifuge tube and centrifuged at 1000g for 6min, the original medium was removed, and 500. Mu.L of fresh medium was added to resuspend the cells. The resuspended cells were then screened to remove cell clumps, collected into flow tubes, and prepared for on-machine sorting. A1.5 mL centrifuge tube was prepared and 500. Mu.L of medium was added for collection of positive cells after sorting.
The operation steps of sorting GFP positive cells by using a flow sorting technology are as follows:
a. GFP was excited with 488nm laser, and the blank control cells were then placed on the machine to determine the area of negative cells.
b. The experimental group of cells were sorted on-machine, first by SSC-A and FSC-A to select live cell areas, then by FSC-W and FSC-H and SSC-W and SSC-H to remove adherent cell portions, by PerCP and FITC-A to further remove dead cell autofluorescence portions, by FSC-A and FITC-A to select positive cell areas, finally positive cell sorting and collection were performed (FIG. 4).
c. GFP positive was sorted into 96-well plates, and 1 cell was allocated to each well at most, and after 5-6 days, the cell status and luminescence were observed. The markers formed single cell clones and GFP positive wells. PCR was performed on the cell extract DNA obtained by flow sorting and Junction PCR identification was performed (FIGS. 5 and 6).
First, primer GFP2F was used: 5'-AGAACGGCATCAAGGTGAAC-3' and GFP2R:5'-TGCTCAGGTAGTGGTTGTCG-3' (135 bp of amplified product) identification of monoclonal cells containing GFP expression cassette; then using the trans-left homology arm primers L3F:5'-GACTGACCGCTCTGTTCCTT-3' and L3R:5'-GATCCCGTGCCACCTTCC-3' (amplification product 1240 bp) and cross right homology arm primer R3F:5'-GTGTCTGCAGGCTCAAAGAG-3' and R3R:5'-AGCGTGTTGAATTTGCCAGT-3' (amplified product 1290 bp) and 404 monoclonal cells, GFP-positive clone 42 and Junction PCR-positive clone 10 were identified, the efficiency of site-directed integration without enrichment was 2.5%, and the efficiency of site-directed integration after GFP enrichment was 23.8%.
Example 3 site-directed integration of exogenous genes by Donor-2 or Donor-3
The flow chart of site-directed integration of the exogenous gene is shown in FIG. 7 (Donor-2) and FIG. 8 (Donor-3).
The structure of the Donor-2 vector is shown in FIG. 7, and the vector uses pUC57 as a skeleton, and F-sg1 sequences are inserted into one side of a foreign gene expression cassette (GOI). The structure of the Donor-3 vector is shown in FIG. 8, and the vector takes pUC57 as a framework, and F-sg1 sequences in the same direction are inserted at two sides of a foreign gene expression cassette (GOI).
1. Cell transfection
The day before transfection, the primary porcine embryo fibroblasts were resuscitated into 6cm dishes and when the cells reached 70% -80% confluence, cell transfection was performed. The cell transfection method was based on nuclear transfer instrument electrotransfection using Basic Primary Fibroblasts Nucleofector (Lonza) electrotransfection under Amaxa Nucleofector (Lonza) single well nuclear transfection system.
The specific operation flow is as follows:
a. collecting cells, and adjusting the number of cells to 1×10 6 Tube, 200g centrifuge for 5min, and remove the culture solution as clean as possible.
b. Cells were resuspended by adding 100. Mu.L of electrotransfection reagent, and 5. Mu.g of Cas9/F-sg1 expression vector plasmid and 5. Mu.g of linearized Donor-2 plasmid or Donor-3 were added, and the electrotransfection system (including cells, transfection reagent, plasmid) was slowly added to the electrotransfection cup along the wall to avoid air bubbles and reduce transfection efficiency. The optimal transfection procedure for PEF cells was U-023, where the viability and transfection efficiency of cells were higher than for the other procedures.
c. After transfection was completed, 500. Mu.L of complete medium (20% FBS+DMEM) was added to the electrotechnical flask, the cells were gently blown off and then transferred to a pre-warmed 6cm dish containing 5mL of culture medium at 37℃with 5% CO 2 Culturing in an incubator. After 72h of electrotransfection, the control cells (not transfected) and experimental cells were aspirated off the medium, rinsed twice with PBS, and digested for 2min with an appropriate amount of trypsin. The digested cells were transferred to a 15mL centrifuge tube and centrifuged at 1000g for 6min, the original medium was removed, and 500. Mu.L of fresh medium was added to resuspend the cells. The resuspended cells were then screened to remove cell clumps, collected into flow tubes, and prepared for on-machine sorting. A1.5 mL centrifuge tube was prepared and 500. Mu.L of medium was added for collection of positive cells after sorting.
2. Sorting GFP positives using flow sorting techniques
The specific operation steps are as follows:
a. GFP was excited with 488nm laser, and the blank control cells were then placed on the machine to determine the area of negative cells.
b. The experimental group cells are sorted on an upper machine, firstly, se:Sub>A living cell arese:Sub>A is selected through SSC-A and FSC-A, then, an adhesion cell part is removed through FSC-W and FSC-H and SSC-W and SSC-H, se:Sub>A dead cell autofluorescence part is further removed through PerCP and FITC-A, se:Sub>A positive cell arese:Sub>A is selected through FSC-A and FITC-A, and finally, positive cell sorting and collection are carried out.
c. GFP positive was sorted into 96-well plates, and 1 cell was allocated to each well at most, and after 5-6 days, the cell status and luminescence were observed. The markers formed single cell clones and GFP positive wells. PCR was performed on the GFP positive cell extract DNA obtained by flow sorting, and junction PCR identification was performed.
First, primer GFP2F was used: 5'-AGAACGGCATCAAGGTGAAC-3' and GFP2R:5'-TGCTCAGGTAGTGGTTGTCG-3' (135 bp of amplified product) identification of monoclonal cells containing GFP expression cassette; specific primer targeting was then used for identification, the primer positions are shown in fig. 9 and 10, and the primer sequences are as follows:
the identification result shows that 382 PEF monoclonal cells are screened in the transfected Donor-2 group, 19 clones positive to the Junction PCR have the efficiency of site-directed integration of 5.0%, and all site-directed integration clones are integrated in the forward direction. In the Donor-3 group, 429 clones were co-screened, 18 clones were positive by Junction PCR, and the site-directed integration efficiency was 4.2%. All site-directed integration clones were forward integrated.
In the invention, the structure of the Donor-1 is shown in figure 11, and the Donor-1 integrates the exogenous gene expression frame to the target site of the No. 12 chromosome of the pig at fixed points through the sgRNA, the Cas9 protein and a homologous recombination mechanism of cells, so that the method has the advantages of accurate integration position and integration direction.
The structure of the Donor-2 is shown in figure 12, and the Donor-2 integrates the exogenous gene expression frame to the target site of the No. 12 chromosome of the pig at fixed points through the sgRNA, the Cas9 protein and a non-homologous end connecting mechanism of cells, has a homologous recombination mechanism independent of the homologous recombination mechanism, can realize fixed-point integration in non-dividing cells, and has higher integration efficiency than the homologous recombination mechanism without a screening marker.
The structure of Donor-3 is shown in FIG. 13. Donor-3 also integrates the exogenous gene expression cassette into the target site of the swine chromosome 12 through the sgRNA, cas9 protein and the non-homologous end joining mechanism of cells, but unlike Donor-2, donor-3 has 2F-sg 1 sites, the integration of the vector backbone into the target site can be avoided.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. The pig COL1A1 gene downstream sequence targeting vector based on the CRISPR-Cas9 system is characterized in that the targeting vector is a pX330 vector containing gRNA;
the DNA sequence of the gRNA recognition site is as follows: 5'-ACGACCCTCCAGTTCGCCTAGGG-3', located in the 3' flanking region of the porcine COL1A1 gene.
2. The donor vector I is characterized by comprising a left homology arm of a downstream sequence targeting region of the pig COL1A1 gene, an exogenous gene expression frame and a right homology arm of the downstream sequence targeting region of the pig COL1A1 gene which are connected in sequence; or alternatively, the process may be performed,
comprises a left homologous arm-insulator-exogenous gene expression frame-insulator-pig COL1A1 gene downstream sequence targeting region right homologous arm which are connected in sequence;
wherein the exogenous gene expression cassette comprises a promoter, an exogenous gene sequence and a terminator.
3. The donor vector of claim 2, wherein the nucleotide sequences of the left and right homology arms of the downstream sequence targeting region of the pig COL1A1 gene are set forth in SEQ ID NOs: 6. shown at 7.
4. A donor vector II comprising a gRNA and an exogenous gene expression cassette;
wherein, the DNA sequence of the gRNA recognition site is: 5'-ACGACCCTCCAGTTCGCCTAGGG-3', which is located in the 3' -flanking region of the porcine COL1A1 gene;
the exogenous gene expression cassette comprises a promoter, an exogenous gene sequence and a terminator.
5. Donor vector III comprising a gRNA-foreign gene expression cassette-gRNA linked in sequence;
wherein, the DNA sequence of the gRNA recognition site is: 5'-ACGACCCTCCAGTTCGCCTAGGG-3', which is located in the 3' -flanking region of the porcine COL1A1 gene;
the exogenous gene expression cassette comprises a promoter, an exogenous gene sequence and a terminator.
6. The donor vector according to any of claims 2 to 5, wherein the backbone vector is pUC57.
7. A CRISPR-Cas9 system targeting the 3' flanking region of the pig COL1A1 gene, comprising the targeting vector of claim 1 and the donor vector of any one of claims 2-6.
8. The use of the CRISPR-Cas9 system of claim 7 in pig gene editing.
9. A method for targeted site-directed integration of exogenous genes in 3' flanking regions of pig COL1A1 genes, characterized in that the targeting vector of claim 1 and the donor vector of any one of claims 2-6 are transfected together into pig embryo fibroblasts, and positive clones are obtained by screening.
10. The method according to claim 9, wherein after transfection of porcine embryo fibroblasts and selection for site-directed integration cloning, the porcine with site-directed integration exogenous gene is obtained by somatic cloning and embryo transfer techniques.
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