WO2016197362A1 - Method for specific knockout of swine vwf gene using crispr-cas9 specificity, and sgrna used for specifically targeting vwf gene - Google Patents

Abstract

Provided is a method for using CRISPR-Cas9 specificity to knock out a swine vWF gene, and an sgRNA used for specifically targeting the vWF gene. The target sequence of the sgRNA for specifically targeting the vWF gene conforms to 5'-N(20)NGG-3' sequence rules, N(20) representing 20 consecutive bases and N representing A or T or C or G; the target sequence in the vWF gene is unique and is located at the 5 exon coding regions, or the junction with the adjacent introns, at the N end of the vWF gene.

Description

CRISPR-Cas9 specific knockdown of swine vWF gene and sgRNA for specific targeting of vWF gene Technical field

The invention relates to the field of genetic engineering technology, in particular to the field of gene knockout technology, in particular to a method for specifically knocking out a swine vWF gene by CRISPR-Cas9 and an sgRNA for specifically targeting a vWF gene.

Background technique

Organ transplantation is the most effective treatment for organ failure diseases. To date, nearly one million patients worldwide have survived through organ transplantation. With the aging of the population and advances in medical technology, more and more patients need organ transplant surgery, but the shortage of donor organs severely restricts the development of organ transplant surgery. Taking kidney transplantation as an example, there are as many as 300,000 patients who need kidney transplantation every year in China, and no more than 10,000 donated kidneys for transplantation. Most of the patients die from kidney failure. Relying on post-mortem organ donation can no longer meet the needs of organ transplantation. Genetic engineering of other species to provide organs suitable for human transplantation has become the main way to address the shortage of human donor organs.

At present, according to the evaluation of biosafety, physiological function indicators, economy and protection of rare species, pigs have become the most ideal source of xenobiotic organs. However, there is a huge difference between pigs and humans. Direct transplantation of pig organs into humans produces strong immune rejection. Therefore, genetic engineering of pigs to produce organs suitable for human transplantation has become the ultimate goal of xenotransplantation.

In the process of transplanting pig lungs to primate mites, it was found that pig-derived lungs released a large amount of vWF (von Willebrand factor) after transplantation, while heterologous reactive antibodies in humans and other primates could bind to vWF. Alpha (1,3) Gal epitope and activates primate platelets. vWF is a kind of glycoprotein with large molecular weight and adhesion function, which can mediate platelet adhesion and promote hemostasis of wounds, while defects of vWF will lead to hemorrhage of mucosa and skin. The vWF-mediated protein-protein interaction plays an important regulatory role in the hemostasis process and thrombus formation. In studies of porcine lung-to-sputum transplantation, it was shown that a large number of hetero-reactive antibodies can bind to vWF released from pig lungs and detach from endothelial cells of porcine pulmonary vessels. The xenoreactive antibody/vWF complex activates and aggregates platelets from humans and other primates by recognizing the Gplb receptor on the platelet surface, resulting in acute lung graft disability.

In the ex vivo human blood perfusion experiment of pig lung, the use of platelet GPIIb-IIIa receptor antagonist or platelet receptor GPIb/vWF interaction inhibitor can significantly improve the survival time of the graft. The use of vWF-deficient pig lungs can also effectively reduce the common coagulopathy during swine-pigment xenografts. In addition, Xenografting with vWF-deficient pig lungs also significantly reduced endothelial cell activation. Therefore, knocking out swine vWF by genetic engineering technology is expected to reduce the xenograft disability syndrome in xenografts using defective vWF pig-derived organs, which will make an important contribution to xenotransplantation. The best way to achieve this strategy is to construct genetically modified pigs with missing vWF molecules.

At present, common gene knockout techniques include homologous recombination (HR) technology, Transcription Activator-Like Effector Nuclease (TALEN) technology, Zinc-Finger Nuclease (ZFN) Technology and the recently developed Law Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technique. Due to the inefficient recombination of HR technology (efficiency is only about 10 ^-6 ), the screening of mutants is very time consuming and inefficient, and has gradually been replaced. The cutting efficiency of TALEN technology and ZFN technology can generally reach 20%, but all need to build protein modules that can recognize specific sequences, and the preliminary work is cumbersome and time consuming. The module design of ZFN technology is complex and has a high off-target rate, and its application is limited.

CRISPR is an acquired immune system derived from prokaryotes that performs a function of interfering functions consisting of protein Cas and CRISPR-RNA (crRNA). At present, the system has found three types, of which the second type of Cas9 system is simple in composition and has been actively applied in the field of genetic engineering. Cas9 targeted cleavage of DNA is achieved by the principle of complementary recognition of two small RNAs, cryRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA), to target sequences. The two small RNAs have now been fused into an RNA strand, abbreviated as sgRNA (single guide RNA), which recognizes specific gene sequences and directs Cas9 protein for cleavage. In eukaryotes, non-homologous recombination ends are ligated after DNA cleavage, resulting in frameshift mutations that ultimately lead to functional knockout of the gene.

Compared with the above three technologies, the CRISPR technology is simple in operation, high in screening efficiency, and capable of achieving accurate targeted cutting. Therefore, knocking out the vWF gene by CRISPR technology can greatly improve the screening efficiency of vWF-deficient cells and genetically engineered pigs. However, the key technical challenge of this path is to design and prepare precisely targeted sgRNAs, because the targeting accuracy of genes is highly dependent on sgRNA target sequences, and the successful design of precisely targeted sgRNAs becomes a key technical issue for knocking out target genes. The present invention is intended to solve this technical problem and thereby provide a solid foundation for knocking out the vWF gene.

Summary of the invention

The object of the present invention is to provide a method for CRISPR-Cas9 specific knockout of the swine vWF gene and an sgRNA for specifically targeting the vWF gene.

According to a first aspect of the invention, the invention provides a CRISPR-Cas9 specific knockout porcine vWF group Because of the sgRNA used to specifically target the vWF gene, the sgRNA has the following characteristics:

(1) The target sequence of the sgRNA on the vWF gene conforms to the sequence alignment rule of 5'-N(20)NGG-3', wherein N(20) represents 20 consecutive bases, wherein each N represents A or T Or C or G, a rule-compliant target sequence may be located in the sense strand or the antisense strand;

(2) the target sequence of the sgRNA on the vWF gene is located in the exon coding region of the vWF gene;

(3) The target sequence of the sgRNA on the vWF gene is unique.

As a preferred embodiment of the present invention, the above target sequence is the sequence shown by any one of SEQ ID NOS: 1 to 58 in the Sequence Listing.

As a preferred embodiment of the present invention, the above target sequence is the sequence shown by SEQ ID NO: 1 in the Sequence Listing.

According to a second aspect of the invention, the invention provides a method for CRISPR-Cas9 specific knockout of a swine vWF gene, the method comprising the steps of:

(1) The 5'-end of the target sequence of the sgRNA described in the first aspect is added to the sequence for forming the cohesive end, and the forward oligonucleotide sequence is synthesized; the target sequence of the sgRNA described in the first aspect The opposite ends of the corresponding complementary sequences are added with appropriate sequences for forming sticky ends, and the reverse oligonucleotide sequence is synthesized; the synthesized forward oligonucleotide sequence is annealed to the reverse oligonucleotide sequence, To form a double-stranded oligonucleotide having a sticky end;

(2) ligating the above double-stranded oligonucleotide into a linearized expression vector carrying the Cas9 gene to obtain an expression vector carrying the sgRNA oligonucleotide and the Cas9 gene containing the corresponding target sequence, transforming the competent bacteria, and screening Identify the correct positive clones, and shake the positive clones and extract the plasmid;

(3) packaging the pseudotyped lentivirus carrying the sgRNA targeting the vWF gene and Cas9 with the expression vector carrying the sgRNA oligonucleotide and the Cas9 gene, the packaging plasmid and the packaging cell line;

(4) infecting the target cell with the pseudotyped lentivirus, and further culturing; then collecting the infected target cell, and amplifying the gene fragment containing the target sequence by using the genomic DNA as a template, and undergoing denaturation, renaturation and enzymatic cleavage, The knockout of the vWF gene was determined.

As a preferred embodiment of the present invention, the above expression vector is a vector of the sequence shown by SEQ ID NO: 59 in the Sequence Listing.

As a preferred solution of the present invention, the above method comprises the following steps:

(1) A forward oligonucleotide sequence is synthesized by adding a CACCG sequence to the 5'-end of the target sequence of the sgRNA of the first aspect; the target sequence corresponding to the target sequence of the sgRNA of the first aspect is The 5'-end plus the AAAC sequence and the 3'-end plus C, the reverse oligonucleotide sequence is synthesized; the synthesized forward oligonucleotide sequence is annealed and renatured with the reverse oligonucleotide sequence, Forming a double-stranded oligo with sticky ends Polynucleotide

(2) The above double-stranded oligonucleotide is ligated into a linearized vector obtained by digesting the expression vector lentiCRISPR v2 of the sequence shown by SEQ ID NO: 59 in the sequence listing by BsmB I restriction endonuclease to obtain a sgRNA. The recombinant expression vector lentiCRISPR v2-vWF of the oligonucleotide was transformed into competent bacteria, and the correct positive clone was screened, and the positive clone was shaken and the plasmid was extracted;

(3) using the above expression vector lentiCRISPR v2-vWF, packaging plasmid and packaging cell line to package a pseudotype lentivirus carrying both sgRNA and Cas9 targeting the vWF gene;

(4) Infecting the target cell with the above-mentioned CRISPR pseudotype lentivirus, and further culturing; then collecting the infected target cell, and amplifying the gene fragment containing the target sequence by using genomic DNA as a template, and undergoing denaturation, renaturation and restriction enzyme digestion To determine the knockout of the vWF gene.

As a preferred embodiment of the present invention, the above packaging plasmid is plasmid pLP1, plasmid pLP2 and plasmid pLP/VSVG; and the above packaging cell line is HEK293T cells.

As a preferred embodiment of the present invention, the above target cells are porcine PIEC cells.

As a preferred embodiment of the present invention, the gene fragment comprising the target sequence is amplified by using the genomic DNA as a template, and the knockdown of the vWF gene is determined by denaturation, renaturation and enzymatic cleavage, specifically:

(a) amplifying a vWF gene fragment comprising the target sequence of the above sgRNA with the genomic DNA of the target cell infected with the virus as a template, and amplifying the genome of the wild-type cell not infected with the same primer with the same primer DNA

(b) purifying the amplified vWF gene fragment, and then mixing the vWF gene fragment from the virus-infected target cell with the vWF gene fragment derived from the wild-type cell, heating and denaturation, and renaturation to form a hybrid DNA molecule;

(c) cutting the renatured hybrid DNA molecule with a Cruiser enzyme;

(d) Electrophoresis detection of the digested product, detection of the target sequence-mediated vWF gene knockout effect.

According to a third aspect of the present invention, the present invention provides a recombinant expression vector lentiCRISPR v2-vWF for use in a method for CRISPR-Cas9 specific knockout of a swine vWF gene, the sequence of the backbone vector of the recombinant expression vector being SEQ ID NO: ID NO: 59; the target sequence carried, such as the target sequence of the sgRNA of the first aspect, is preferably the target sequence shown by SEQ ID NO: 1 in the sequence listing.

According to a fourth aspect of the present invention, the present invention provides the use of the sgRNA according to the first aspect or the recombinant expression vector lentiCRISPR v2-vWF according to the third aspect, in the method of CRISPR-Cas9 specific knockout of the swine vWF gene.

The present invention specifically knocks out the swine vWF gene against CRISPR-Cas9 and successfully finds a specific target The sgRNA of the vWF gene, the sgRNA of the invention is used in the method of CRISPR-Cas9 specific knockout of the swine vWF gene, and the swine vWF gene can be rapidly, accurately, efficiently and specifically knocked out, and the vWF gene knocking is effectively solved. In addition to the technical problems of long pig cycle and high cost.

DRAWINGS

Figure 1 is a plasmid map of the vector plasmid lentiCRISPR v2 used in the examples of the present invention;

Figure 2 is a plasmid map of the packaging plasmid pLP1 used in the embodiment of the present invention;

Figure 3 is a plasmid map of the packaging plasmid pLP2 used in the examples of the present invention;

Figure 4 is a plasmid map of the packaging plasmid pLP/VSVG used in the examples of the present invention;

5 is a diagram showing the results of electrophoresis detection of the gene knock-out effect of the target sequence in the enzyme digestion assay according to the embodiment of the present invention, wherein M represents DNA Marker, and 1 represents the targeted cleavage effect of the No. 1 target sequence in Table 1 on the vWF gene, WT The result of the PCR product of the wild type cell which has not been subjected to viral infection and Cas9 cleavage is the result of a Bruiser digestion test, and the arrow indicates a small fragment obtained by cutting with a Cruiser enzyme.

detailed description

The technical solutions of the present invention are further described below in conjunction with the accompanying drawings and specific embodiments. The drawings and specific examples are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the materials used are all commercially available.

The test materials and reagents involved in the following examples: lentiCRISPR v2 plasmid was purchased from Addgene, packaging plasmids pLP1, pLP2 and pLP/VSVG were purchased from Invitrogen, and packaging cell line HEK293T cells were purchased from the American Model Culture Collection (ATCC). PIEC cells were purchased from the Chinese Academy of Sciences cell bank, DMEM medium, Opti-MEM medium and fetal bovine serum FBS were purchased from Gibco, and Lipofectamine 2000 was purchased from Invitrogen.

The molecular biology experimental methods not specifically described in the following examples are all carried out according to the specific method described in the book "Molecular Cloning Experimental Guide" (third edition) J. Sambrook, or according to the kit and product manual. .

The general technical solution of the present invention includes the following five parts:

I. Selection and design of Sus scrofa (pig) vWF gene sgRNA target sequence

1. Selection of sgRNA target sequence of vWF gene:

A suitable 20 bp oligonucleotide sequence was searched for as a target sequence in the exon region of the vWF gene.

2. The sgRNA target sequence design of the vWF gene:

The above target sequence and complementary sequence are separately added to the linker to form a forward oligonucleotide sequence and a reverse oligonucleotide sequence.

Second, the construction of the vWF gene CRISPR carrier

1. synthesizing the above forward oligonucleotide sequence and reverse oligonucleotide sequence, renaturation to form a double-stranded DNA fragment having a sticky end (ie, a double-stranded target sequence oligonucleotide, which may also be referred to as a double-stranded oligonucleotide Polynucleotide).

2. Construction of CRISPR-sgRNA expression vector:

The above double-stranded DNA fragment was constructed into a target vector (e.g., lenti CRISPR V2, the plasmid map of which is shown in Figure 1) to form a lentiviral CRISPR vector such as lenti CRISPR SP2-vWF.

Third, obtain pseudotyped lentivirus expressing vWF sgRNA

A CRISPR pseudotyped lentivirus expressing a vWF sgRNA was produced using a packaging plasmid, a packaging cell line, and a lentiviral CRISPR vector.

Fourth, infect the target cells and detect the knockout effect of vWF gene

1. Lentivirus infection of the target cell:

A pseudotyped lentivirus such as lentiCRISPR v2-vWF is added to the cell culture medium of interest for infection and further culture.

2. Detection of vWF gene knockout effect:

The target cells are collected, and the gene fragment containing the target sequence is amplified by using genomic DNA as a template, and the knockdown of the vWF gene is determined by denaturation, renaturation and restriction enzyme digestion.

V. Selection and identification of vWF knockout monoclonal

1. For a target cell population with a defined knockout effect, a number of single cell derived cell lines are isolated by dilution and monoclonal culture.

2. Identification of monoclonal vWF knockouts.

The technical solutions of the present invention and the beneficial effects thereof will be described in detail below by way of examples.

Example 1. Selection and design of Sus scrofa (pig) vWF gene sgRNA target sequence

The target sequence determines the targeting specificity of the sgRNA and the efficiency of the Cas9-cleaving gene of interest. Therefore, efficient and specific target sequence selection and design are prerequisites for the construction of sgRNA expression vectors.

1. Selection of sgRNA target sequence of vWF gene

For the vWF gene, the following principles should be followed in the selection of target sequences:

(1) Find a target sequence conforming to the 5'-N(20)NGG-3' rule in the exon coding region of the vWF gene, wherein N(20) represents 20 contiguous bases, wherein each N represents A or T Or C or G, a rule-compliant target sequence may be located in the sense strand or the antisense strand;

(2) selecting the sequence of the exon coding region, such that the cleavage of the coding region sequence results in the functional knockout of the vWF gene, and the residual truncated sequence does not form a functional protein;

(3) If there are multiple splicing bodies, the selection is performed in the shared exon coding region, and the exon coding region sequence near the N-terminus is selected for the vWF gene to satisfy the condition;

(4) Using the online sequence analysis tool (http://crispr.mit.edu/) to analyze the homology of the above target sequences in the pig genome, discard the target sequences with significant homologous sequences, and further select according to the scores. The target sequence is unique on the vWF gene.

Based on the above principles, the target sequence set shown in Table 1 was selected.

Table 1 Target sequence collection

Numbering		sequence
			1	GCCCCGCGGGCATGGAGTAC
2	CGCGCTCCTGGAGTATGCCC
		3	GACGCTTGGCAAATCTTCGA
4	ATCTTGGAAGTCCCCGATGA
		5	GGTTCCTGTCAGGCTTGCGA
6	TGGTTCCTGTCAGGCTTGCG
		7	TGATGAAATTGCTTCCGAAG
8	ATCATCGGGGACTTCCAAGA
		9	AGCACCCTCGCAAGCCTGAC
10	GCTGTACATGCTCTGATCAA
		11	ACACTCCTTCTCCATCATCG
12	GAAGTCCCCGATGATGGAGA
		13	CAGAGCATGTACAGCTTTGC
14	AGGAAGCTGCAGTTACCTCC
		15	CTCTGTGGAGAAGAGACGCT
16	GCTTCCGAAGAGGCTGCATC
		17	TGCGAGGGTGCTGCTTGCCC
18	AGCTGCAGTTACCTCCTGGC

19	GGCCCACTCTCTTGCCATCT
		20	CACAAAGCCGTAGGCCTCGC
twenty one	GTTGCCGCTCCCATCAATTC
		twenty two	CAGCGAGGCCTACGGCTTTG
twenty three	AATTCTGGCCACAAAGCCGT
		twenty four	GTACCACACGCTGTCCAGCG
25	ACGCTGTCCAGCGAGGCCTA
		26	CCCTTTGCTGGCATAGGGCG
27	TGTCAATGGCACCGTGCTGC
		28	GATACTTCAACAAGACCTGC
29	GTCAATGGCACCGTGCTGCA
		30	CATAGGGCGTGGAGATGCTT
31	CGGTGCCATTGACAAACACA
		32	TGAAGTATCTGTCTGACAGC
33	TCAATGGCACCGTGCTGCAG
		34	TACAGCCCTTTGCTGGCATA
35	TGCCAGCAAAGGGCTGTATC
		36	GGAGATGTTGCACGAGATGC
37	AAAGTCGCCACAGAGCCCGC
		38	GGCAAAGCTGTAGGGGTCCG
39	GAGATGTTGCACGAGATGCT
		40	AGCTGTAGGGGTCCGAGGTC
41	AGGGCATGCTGCTCTACGGC
		42	CCCTGTACTCCATGCCCGCG
43	GGGCATACTCCAGGAGCGCG

44	CCGCGCTCCTGGAGTATGCC
		45	CGGGCATACTCCAGGAGCGC
46	CCCCGCGGGCATGGAGTACA
		47	CACAGGCCCGGGCATACTCC
48	CATGCTGCTCTACGGCTGGA
		49	CCGGGCATACTCCAGGAGCG
50	GGTGGACCCCGAGCCTTTCG
		51	TCCCTGTACTCCATGCCCGC
52	TCCATGCCCGCGGGGCAGTC
		53	TTCTCACACAGGGCCACGAA
54	ACAGGGCCACGAAAGGCTCG
		55	CTCCCTGTACTCCATGCCCG
56	TACTCCAGGAGCGCGGGGCA
		57	CGTAGAGCAGCATGCCCTGC
58	CGAAAGGCTCGGGGTCCACC

2. The sgRNA target sequence design of the vWF gene:

(1) Using the lenti CRISPR PR2 plasmid as an expression vector, the CACCG sequence was added to the 5'-end of the above N(20) target sequence to form a forward oligonucleotide sequence according to the characteristics of the lenti CRISPR SP2 plasmid:

5’-CACCGNNNNNNNNNNNNNNNNNNNN-3’;

(2) adding a sequence at both ends of the reverse complement of the above N(20) target sequence to form a reverse oligonucleotide sequence:

5'-AAACNNNNNNNNNNNNNNNNNNNNC-3';

The forward oligonucleotide sequence and the reverse oligonucleotide sequence can be complementary to form a double-stranded DNA fragment having a sticky end:

5’-CACCGNNNNNNNNNNNNNNNNNNNN-3’

3'-CNNNNNNNNNNNNNNNNNNNNCAAA-5'.

Example 2: Construction of sgRNA expression vector of vWF gene

Synthetic DNA insert

(1) Synthesis of forward and reverse oligonucleotide sequences of the above design

Oligonucleotide sequences can be specifically synthesized by commercial companies (such as Invitrogen) according to the sequences provided. This example and the following examples investigate the knockout effect of the target sequence shown in the first sequence listed in Table 1 on the vWF gene.

The forward oligonucleotide sequence and the reverse oligonucleotide sequence corresponding to the No. 1 target sequence are as follows:

GACCGGCCCCGCGGGCATGGAGTAC (SEQ ID NO: 60);

AAACGTACTCCATGCCCGCGGGGCC (SEQ ID NO: 61).

The corresponding forward and reverse oligonucleotide sequences are annealed and renatured to form a double-stranded DNA fragment having sticky ends.

The reaction system (20 μL) is as follows:

Forward oligonucleotide (10 μM): 1 μL

Reverse oligonucleotide (10 μM): 1 μL

10×PCR buffer: 2 μL

ddH ₂ O: 16 μL

The above reaction system was placed in a PCR machine, and the reaction was carried out in accordance with the following procedure.

Reaction procedure:

95 ° C, 5 min;

80 ° C, 5 min;

70 ° C, 5 min;

60 ° C, 5 min;

50 ° C, 5 min;

Naturally reduced to room temperature.

2. Construction of sgRNA expression vector

(1) The target vector lentiCRISPR v2 plasmid (the sequence of which is shown in SEQ ID NO: 59 in the Sequence Listing) was digested with BsmB I restriction endonuclease.

Prepared according to the following reaction system:

Lenti CRISPR PR2 Plasmid: 1μg

10× digestion buffer: 2 μL

BsmB I restriction enzyme: 2 μL

Supplement ddH ₂ O to a total volume of 20 μL

The digestion reaction system was placed at 37 ° C for 4 h.

(2) Electrophoretic separation and purification of vector fragments

After the end of the digestion, the digestion mixture was separated by agarose gel electrophoresis, and the vector fragment (about 12 kb) was selected for cleavage and recovered by a DNA gel recovery column.

(3) linking the synthesized double-stranded DNA fragment to the vector main fragment and transforming Escherichia coli

The double-stranded DNA fragment obtained by renaturation is linked with the recovered vector fragment, and is prepared according to the following reaction system:

LentiCRISPR v2 vector fragment: 100ng

Double-stranded DNA fragment: 200ng

T4 ligase: 1 μL

T4 connection reaction buffer: 1μL

Supplement ddH ₂ O to a total volume of 10 μL

The ligation mixture was reacted at 25 ° C for 2 h.

After the reaction, the ligation mixture was transformed into E. coli DH5α strain: 100 μL of E. coli DH5α competent cells were added to the ligation mixture, and incubated on ice for 30 min; the mixture was placed in a 42 ° C water bath, heat shocked for 90 s, and then placed on ice to cool; 100 μL of LB medium was added and incubated at 37 ° C for 20 min on a shaker; the mixture was coated with Amp LB plates and incubated at 37 ° C for 14 h.

(4) Identification of correct transformed clones

Several colonies were selected from Amp LB plates for expansion culture, and plasmids were extracted for restriction enzyme digestion. Pick the clones that are likely to be correct for sequencing and verify that the insert is correct. The correct lenti CRISPR PR2-vWF vector clone was seeded.

Example 3, obtaining a pseudotype lentivirus expressing vWF sgRNA

1. Material preparation

Amplify and extract the packaging plasmids pLP1, pLP2 and pLP/VSVG (purchased from Invitrogen, the maps are shown in Figure 2, Figure 3 and Figure 4, respectively); amplify and extract the vector plasmid lentiCRISPR v2-vWF; culture packaging cells HEK293T cells (purchased from ATCC); DMEM medium, Opti-MEM medium and fetal bovine serum FBS (purchased from Gibco); Lipofectamine 2000 (purchased from Invitrogen); HEK293T cells cultured in 37 ° C culture environment containing 5% CO ₂ The medium was DMEM medium containing 10% FBS.

2. Transfection and virus packaging

Day 1: Passage of the packaging cell line HEK293T to a 10 cm dish, approximately 30% confluence;

Day 2: Transfection was performed according to the following formula when HEK293T reached 80% confluency:

Formulation of Mixture 1, comprising:

lentiCRISPR v2-vWF: 6μg

pLP1: 6μg

pLP2: 6μg

pLP/VSVG: 3μg

Opti-MEM: 500 μL.

Formulation of Mixture 2, comprising:

Lipofectamine 2000: 30 μL

Opti-MEM: 500 μL.

After standing for 5 min, mixture 1 and mixture 2 were mixed to form a transfection mixture and allowed to stand for 20 min.

The HEK293T medium was changed to serum-free DMEM medium, and the transfection mixture was added. After incubation at 37 ° C for 8 hours, the cells were replaced with 20% FBS DMEM medium, and the culture was continued.

3. Virus collection and preservation

Day 3: The virus-containing HEK293T medium supernatant was collected 48 h after transfection, filtered through a 0.45 μm filter, dispensed, and stored at -80 °C.

Example 4, infecting the target cell and detecting the knockout effect of the target sequence

1. Material preparation

The target cell line was porcine hip arterial endothelial cells PIEC (purchased from the Chinese Academy of Sciences cell bank); DMEM medium and fetal bovine serum FBS (purchased from Gibco); target sequence (sequence 1) lentiCRISPR v2-vWF pseudotype lentivirus; The PIEC cells were cultured in a 37 ° C culture environment containing 5% CO ₂ in DMEM medium containing 10% FBS.

2. Lentivirus infection of the target cell

Day 1: Passage cells of interest to 6-well plates at approximately 20% fusion density. Each virus requires a 6-well and requires an efficiency of 6 wells.

Day 2: 1 mL of lentiCRISPR v2-vWF pseudotype lentiviral supernatant and 1 mL of DMEM medium were added to the target cells at approximately 40% confluency. Efficiency comparisons do not require the addition of lentiviruses.

Day 3: After 24 hours of infection, the virus-containing medium was removed, replaced with normal medium, and puromycin was added to a final concentration of 2 μg/mL. The control sample without the virus infection was also added with puromycin as a control and cultured for 48 hours.

3. Detection and culture of cell infection efficiency

Day 5: Uninfected efficacious control cells should all be apoptotic (>95%) under the action of puromycin. According to the apoptosis of infected lentiviral cells, the infection efficiency of cells can be determined, and the infection efficiency of 90% or more can be achieved (apoptosis rate <10%). If necessary, the virus supernatant can be concentrated or diluted to be infected to achieve appropriate infection efficiency.

After infection with puromycin, uninfected cells undergo apoptosis. The cells of interest were re-passaged and replaced with normal medium for 48 h.

4. Detection of vWF gene knockout effect

(1) Design upstream and downstream primers to amplify the vWF gene fragment, wherein the upstream and downstream primer sequences are as follows:

TCTGAGCACCACATGGCTTG (SEQ ID NO: 62);

ACACCAGCTCAATCCTGATC (SEQ ID NO: 63).

(2) Collect some target cells and extract genomic DNA using the promega genomic DNA kit. Simultaneous extraction of genomic DNA from wild-type cells of interest.

(3) Amplifying a vWF gene fragment (including an infected mutant sample and a wild type sample) containing the target sequence using genomic DNA as a template.

The amplification reaction system (20 μL) was as follows:

Upstream primer (10μM): 1μL

Downstream primer (10μM): 1μL

2×PCR Mix: 10 μL

Genomic DNA: 100ng

The above reaction system was prepared, placed in a PCR machine, and reacted according to the following procedure.

Reaction procedure:

95 ° C, 3 min

95 ° C, 30 s

58°C, 20s

72°C, 20s

72 ° C, 3 min;

The second to fourth steps are repeated for 35 cycles.

(4) Electrophoresis detection of PCR products and recovery and purification

(5) The purified DNA fragments are separately denatured and renatured to form hybrid DNA molecules (including mutant samples and wild-type samples).

The reaction system is as follows:

Genomic PCR fragment: 200ng

5× reaction buffer: 2 μL

The reaction system has a total of 9μL

The above reaction system was prepared, placed in a PCR machine, and reacted according to the following procedure.

Reaction procedure:

95 ° C, 5 min;

80 ° C, 5 min;

70 ° C, 5 min;

60 ° C, 5 min;

50 ° C, 5 min;

Naturally reduced to room temperature.

(6) Cutting the renatured hybrid DNA (including mutant samples and wild-type samples) with Cruiser enzyme

1 μL of Cruiser enzyme was added to the denatured, renatured reaction mixture and incubated at 45 ° C for 20 min.

(7) Electrophoresis detection of the digested product, detection of the target sequence-mediated vWF gene knockout effect.

The digested DNA fragment was subjected to electrophoresis on a 2% agarose gel, 100 V, 25 min. The cutting condition of the target fragment is determined, and the gene knocking effect of the target sequence is judged.

The cleavage recognition of mutant DNA is based on the principle that infected cells express sgRNA and Cas9. Genomic DNA, if sgRNA-mediated Cas9 protein-targeted cleavage, is introduced to introduce mutations near the cleavage site (wild-type becomes mutant). Since the wild type and the mutant sequence do not match at this position, the hybrid molecule in which the wild type DNA amplified by the template and the mutant DNA undergoes renaturation will generate a local loop structure. The latter can be recognized and cleaved by the Cruiser enzyme, resulting in the hybrid DNA molecule being cleaved into small fragments.

As a result, as shown in Fig. 5, a small fragment was not detected in the PCR product of the wild-type cell which was not infected with the virus; and the sequence 1 was able to effectively target the vWF gene to produce a cleavage, and thus the presence of a small fragment was detected, indicating that the sequence 1 can be used as a CRISPR -Cas9 specifically knocks out the target sequence of the swine vWF gene.

Example 5: Selection and identification of vWF knockout monoclonal

1. Selection of monoclonal (based on sequence 1 target sequence)

(1) The partially infected cell population was passaged, and 100 single cells were transferred to a 10 cm dish for culture.

(2) After about 10 days of culture, a considerable amount of the monoclonal grows to a level visible to the naked eye.

(3) A separate clone was scraped with a pipette tip, and the cells were transferred to a 24-well plate, and one well was corresponding to each well.

(4) After about one week of culture, some clones are grown to a sufficient number for further identification.

2. Identification of monoclonal vWF knockouts

(1) Collect the monoclonal and wild-type cells to be examined and extract the genomic DNA separately.

(2) A vWF gene fragment of monoclonal and wild-type cells is amplified according to the aforementioned method, and the amplified gene fragment comprises an sgRNA target sequence.

(3) Mixing an equal amount of the monoclonal PCR fragment with the wild type PCR fragment, heating and denaturation, and renaturation to form a hybrid DNA molecule.

(4) The annealed hybrid DNA was cleaved with a Cruiser enzyme and incubated at 45 ° C for 20 min.

(5) Electrophoresis detection of the digested product, and whether the monoclonal has an effective mutation is determined according to whether or not there is a cleavage fragment.

The results showed that the lentiCRISPR v2-vWF pseudotyped lentivirus infection target cell based on the target sequence shown in SEQ ID NO:1, 20 randomly selected from 100 single cells were detected by Cruiser enzyme electrophoresis, and 17 of them were single. Cloning can detect small fragments, indicating that gene knockout occurs, and the knockout efficiency can reach more than 85%, indicating that the target sequence shown in sequence 1 has a high target for knocking out the vWF gene.

The above is a further detailed description of the present invention in connection with the specific embodiments, and the specific embodiments of the present invention are not limited to the description. A number of simple derivations or substitutions may be made by those skilled in the art without departing from the inventive concept.

Claims (10)

1. An sgRNA for specifically targeting the vWF gene in the CRISPR-Cas9 specific knockout porcine vWF gene, characterized by:

   (1) The target sequence of the sgRNA on the vWF gene conforms to the sequence alignment rule of 5'-N(20)NGG-3', wherein N(20) represents 20 consecutive bases, wherein each N represents A or T or C or G, a rule-compliant target sequence may be located in the sense strand or the antisense strand;

   (2) the target sequence of the sgRNA on the vWF gene is located in the exon coding region of the vWF gene;

   (3) The target sequence of the sgRNA on the vWF gene is unique.
2. The sgRNA for specifically targeting the vWF gene according to claim 1, wherein the target sequence is the sequence shown by any one of SEQ ID NOS: 1 to 58 in the Sequence Listing.
3. The sgRNA for specifically targeting the vWF gene according to claim 1, wherein the target sequence is the sequence shown by SEQ ID NO: 1 in the Sequence Listing.
4. A method for specifically knocking out a swine vWF gene by CRISPR-Cas9, characterized in that the method comprises the following steps:

   (1) The 5'-end of the target sequence of the sgRNA according to any one of claims 1 to 3, which is a sequence for forming a cohesive end, which is synthesized to obtain a forward oligonucleotide sequence; Any one of the ends of the complementary sequence corresponding to the target sequence of the sgRNA, plus a suitable sequence for forming a cohesive end, to synthesize a reverse oligonucleotide sequence; the forward oligonucleotide to be synthesized The sequence is annealed and renatured with the reverse oligonucleotide sequence to form a double-stranded oligonucleotide having a sticky end;

   (2) ligating the double-stranded oligonucleotide into a linearized expression vector carrying the Cas9 gene to obtain an expression vector carrying the sgRNA oligonucleotide containing the corresponding target sequence and the Cas9 gene, and transforming the competent bacteria, The correct positive clone was identified by screening, and the positive clone was shaken and the plasmid was extracted;

   (3) packaging the pseudotyped lentivirus carrying the sgRNA targeting the vWF gene and Cas9 with the expression vector carrying the sgRNA oligonucleotide and the Cas9 gene, the packaging plasmid and the packaging cell line;

   (4) infecting the target cell with the pseudotype lentivirus, and further culturing; then collecting the infected target cell, and amplifying the gene fragment containing the target sequence by using genomic DNA as a template, and undergoing denaturation, renaturation and enzymatic Cut and determine the knockout of the vWF gene.
5. The CRISPR-Cas9-specific knock-out porcine vWF gene according to claim 4, wherein the expression vector is a vector of the sequence of SEQ ID NO: 59 in the Sequence Listing.
6. A method for specifically knocking out a swine vWF gene by a CRISPR-Cas9 according to claim 4 or 5, wherein the method comprises the steps of:

   (1) The 5'-end of the target sequence of the sgRNA according to any one of claims 1 to 3 plus CACCG The sequence is synthesized to obtain a forward oligonucleotide sequence; the 5'-end of the complementary sequence corresponding to the target sequence of the sgRNA according to any one of claims 1 to 3 is added to the AAAC sequence, and the 3'-end is added with C, Synthesizing a reverse oligonucleotide sequence; annealing and renaturation of the synthesized forward oligonucleotide sequence to a reverse oligonucleotide sequence to form a double-stranded oligonucleotide having a sticky end;

   (2) The double-stranded oligonucleotide is ligated into a linearized vector obtained by digesting the expression vector lentiCRISPR v2 of the sequence shown by SEQ ID NO: 59 in the sequence listing by BsmB I restriction endonuclease to obtain a vector. The recombinant expression vector lentiCRISPR v2-vWF of sgRNA oligonucleotide is transformed into competent bacteria, and the correct positive clone is identified by screening, and the positive clone is shaken and the plasmid is extracted;

   (3) using the expression vector lentiCRISPR v2-vWF, packaging plasmid and packaging cell line to package a pseudotype lentivirus carrying both sgRNA and Cas9 targeting the vWF gene;

   (4) infecting the target cell with the pseudotype lentivirus, and further culturing; then collecting the infected target cell, and amplifying the gene fragment containing the target sequence by using genomic DNA as a template, and undergoing denaturation, renaturation and enzymatic Cut and determine the knockout of the vWF gene.
7. The CRISPR-Cas9-specific knockout porcine vWF gene according to claim 6, wherein the packaging plasmid is plasmid pLP1, plasmid pLP2 and plasmid pLP/VSVG; and the packaging cell line is HEK293T cells.
8. The method according to claim 6, wherein the target cell is a porcine PIEC cell;

   The gene fragment comprising the target sequence is amplified by using the genomic DNA as a template, and the knockdown of the vWF gene is determined by denaturation, renaturation and enzymatic cleavage, specifically:

   (a) amplifying a vWF gene fragment containing the target sequence of the sgRNA with the genomic DNA of the target cell infected with the virus as a template, and amplifying the wild-type cell of the uninfected virus with the same primer using the upstream and downstream primers of the vWF gene. Genomic DNA;

   (b) purifying the amplified vWF gene fragment, and then mixing the vWF gene fragment from the virus-infected target cell with the vWF gene fragment derived from the wild-type cell, heating, denaturation, and renaturation to form a hybrid DNA molecule;

   (c) cutting the renatured hybrid DNA molecule with a Cruiser enzyme;

   (d) Electrophoresis detection of the digested product, detection of the target sequence-mediated vWF gene knockout effect.
9. The recombinant expression vector lentiCRISPR v2-vWF used in the method of CRISPR-Cas9 specific knockout of the swine vWF gene, wherein the sequence of the backbone vector of the recombinant expression vector is as shown in SEQ ID NO: 59 in the sequence listing. The target sequence carried by the sgRNA of any one of claims 1-3 The target sequence is preferably the target sequence shown by SEQ ID NO: 1 in the Sequence Listing.
10. Use of the sgRNA according to any one of claims 1 to 3 or the recombinant expression vector lentiCRISPR v2-vWF according to claim 9 in a method of CRISPR-Cas9 specific knockout of a swine vWF gene.

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