CN105492609A - Method for CRISPR-Cas9 specific knockout of pig GGTA1 gene and sgRNA for specific targeted GGTA1 gene - Google Patents

Abstract

The invention discloses a method for CRISPR-Cas9 specific knockout of a pig GGTA1 gene and sgRNA for a specific targeted GGTA1 gene. The target sequence of the sgRNA for the specific targeted GGTA1 gene on the GGTA1 gene is in accordance with the sequence arrangement rule of 5'-N(20)NGG-3', wherein N(20) represents 20 continuous bases, and each N represents A or T ot C or G; the target sequence on the GGTA1 gene is positioned at a junction of 5 exon coding areas and/or adjacent introns of the N-end of the GGTA1 gene; and the target sequence on the GGTA1 gene is unique. According to the method for CRISPR-Cas9 specific knockout of the pig GGTA1 gene via sgRNA, specific knockout of the pig GGTA1 gene can be rapidly and accurately realized with high efficiency, and the problems of long period and high cost for constructing the knockout of the pig GGTA1 gene can be effectively solved.

Description

CRISPR-Cas9 method for specifically knocking out pig GGTA1 gene and sgRNA for specifically targeting GGTA1 gene

technical field

The invention relates to the technical field of genetic engineering, in particular to the technical field of gene knockout, in particular to a method for specifically knocking out the pig GGTA1 gene by CRISPR-Cas9 and an sgRNA for specifically targeting the GGTA1 gene.

Background technique

Organ transplantation is the most effective treatment for diseases of organ failure. So far, nearly one million patients around the world have extended their lives through organ transplantation. With the aging of the population and the advancement of medical technology, more and more patients need organ transplantation, but the shortage of donor organs seriously restricts the development of organ transplantation. Taking kidney transplantation as an example, as many as 300,000 patients need kidney transplantation in my country every year, but no more than 10,000 donated kidneys can be used for transplantation, and most patients die of kidney failure. Relying on organ donation after death can no longer meet the needs of organ transplantation. Transforming other species through genetic engineering to provide organs suitable for human transplantation has become the main way to solve the shortage of human donor organs.

At present, pigs have become the most ideal source of xenogeneic organs according to various evaluations such as biological safety, physiological function indicators, economical efficiency, and protection of rare species. However, there are huge differences between pigs and humans, and directly transplanting pig organs into humans will cause strong immune rejection. Therefore, the transformation of pigs through genetic engineering to produce organs suitable for human transplantation has become the ultimate goal of xenotransplantation.

Immunological studies have found that pig cells express a carbohydrate compound: α-1,3-galactose compound (α-1,3-Gal). Its synthesis depends on α-galactosyltransferase 1 (alpha-galactosyltransferase1, GGTA1). Human cells are unable to synthesize alpha-1,3-galactose compounds due to the lack of a functional GGTA1 gene. Such compounds widely exist in bacteria and non-primates. Under the stimulation of bacteria in the body, the human body will produce a large number of antibodies against α-1,3-galactose compounds. If a pig's organ is transplanted to a human, hyperacute rejection will occur due to the corresponding antibody, resulting in failure of the transplant. Therefore, there is a need to eliminate α-1,3-galactose compound molecules on the surface of porcine cells to reduce the immunogenicity of xenogeneic donor organs. The accurate and efficient knockout of pig GGTA1 gene is a key step to eliminate the immune rejection caused by α-1,3-Gal.

Currently, common gene knockout technologies include homologous recombination (Homologus Recombination, HR) technology, Transcription Activator-Like Effector Nuclease (TALEN) technology, Zinc-Finger Nuclease (Zinc-Finger Nuclease, ZFN) technology and the recent development The regular clustered interspaced short palindromic repeat (Clustered Regularly Interspaced Short Palindromic Repeat, CRISPR) technology. Due to the low efficiency of recombination (only about 10 ^-6 ), HR technology has been gradually replaced by the time-consuming and inefficient screening of mutants. The cutting efficiency of TALEN technology and ZFN technology can generally reach 20%, but both require the construction of protein modules that can recognize specific sequences, and the preliminary work is cumbersome and time-consuming. The module design of ZFN technology is relatively complex and has a high off-target rate, and its application is limited.

CRISPR is an acquired immune system derived from prokaryotes. The complex that performs the interference function of this system is composed of the protein Cas and CRISPR-RNA (crRNA). At present, three types of this system have been found, among which the second type of Cas9 system has a simple composition and has been actively applied in the field of genetic engineering. The targeted cutting of DNA by Cas9 is achieved through the principle of complementary recognition of two small RNAs—crRNA (CRISPRRNA) and tracrRNA (trans-activatingcrRNA)—to the target sequence. Now two small RNAs have been fused into one RNA chain, referred to as sgRNA (singleguideRNA), which can recognize a specific gene sequence and guide the Cas9 protein to cut. In eukaryotes, non-homologous recombination of end-joining occurs after DNA is cut, resulting in frameshift mutations, which eventually lead to functional knockout of genes.

Compared with the above three technologies, CRISPR technology is simple to operate, has high screening efficiency, and can achieve precise targeted cutting. Therefore, knocking out the GGTA1 gene by CRISPR technology can greatly improve the screening efficiency of α-1,3-Gal deficient cells and genetically engineered pigs. However, the key technical problem of this approach is to design and prepare precisely targeted sgRNA, because the targeting accuracy of genes is highly dependent on the sgRNA target sequence, and whether the precise targeted sgRNA can be successfully designed becomes a key technical issue for knocking out the target gene , the present invention aims to solve this technical problem so as to provide a solid foundation for knocking out the GGTA1 gene.

Contents of the invention

The object of the present invention is to provide a method for specifically knocking out the pig GGTA1 gene by CRISPR-Cas9 and an sgRNA for specifically targeting the GGTA1 gene.

According to the first aspect of the present invention, the present invention provides an sgRNA for specifically targeting the GGTA1 gene in CRISPR-Cas9 specific knockout of the pig GGTA1 gene, the sgRNA has the following characteristics:

(1) The target sequence of the sgRNA on the GGTA1 gene conforms to the sequence arrangement rule of 5'-N(20)NGG-3', where N(20) represents 20 consecutive bases, and each N represents A or T Or C or G, the target sequence that meets the above rules is located on the sense strand or the antisense strand;

(2) The target sequence of the sgRNA on the GGTA1 gene is located in the 5 exons coding region of the N-terminus of the GGTA1 gene, or a part of the target sequence is located in the 5 exons of the N-terminus of the GGTA1 gene, and the rest of the target sequence is located in the N-terminus of the GGTA1 gene The junction of introns, located in adjacent introns;

(3) The target sequence of the sgRNA on the GGTA1 gene is unique.

As a preferred solution of the present invention, the above-mentioned target sequence is a sequence shown in any one of SEQ ID NO: 1-38 in the sequence listing.

As a preferred solution of the present invention, the above-mentioned target sequence is the sequence shown in SEQ ID NO: 1 in the sequence listing.

According to a second aspect of the present invention, the present invention provides a method for using CRISPR-Cas9 to specifically knock out the pig GGTA1 gene, the method comprising the following steps:

(1) Add a sequence for forming a cohesive end to the 5'-end of the target sequence of the sgRNA described in the first aspect, and synthesize a forward oligonucleotide sequence; the target sequence of the sgRNA described in the first aspect Add appropriate sequences for forming cohesive ends to the two ends of the corresponding complementary sequence to synthesize a reverse oligonucleotide sequence; anneal the synthesized forward oligonucleotide sequence to the reverse oligonucleotide sequence and repeat properties, forming double-stranded oligonucleotides with cohesive ends;

(2) The above-mentioned double-stranded oligonucleotide is connected into the expression vector carrying the Cas9 gene of linearization, and the expression vector carrying the sgRNA oligonucleotide and the Cas9 gene containing the corresponding target sequence is obtained, transformed into competent bacteria, and screened Identify the correct positive clones, shake the positive clones, and extract the plasmids;

(3) using the above-mentioned expression vectors, packaging plasmids and packaging cell lines carrying sgRNA oligonucleotides and Cas9 genes to package pseudotyped lentiviruses carrying both sgRNA and Cas9 targeting the GGTA1 gene;

(4) Use the above-mentioned pseudotyped lentivirus to infect the target cells, and further culture; then collect the infected target cells, use their genomic DNA as a template to amplify the gene fragment containing the above-mentioned target sequence, and undergo denaturation, renaturation and enzyme digestion, Determination of the knockout status of the GGTA1 gene.

As a preferred solution of the present invention, the above-mentioned expression vector is a vector of the sequence shown in SEQ ID NO: 39 in the sequence listing.

As a preferred version of the present invention, the above-mentioned method comprises the steps of:

(1) Add a CACCG sequence to the 5'-end of the target sequence of the sgRNA described in the first aspect, and synthesize a forward oligonucleotide sequence; the complementary sequence corresponding to the target sequence of the sgRNA described in the first aspect Add AAAC sequence to the 5'-end and C to the 3'-end to synthesize the reverse oligonucleotide sequence; anneal and anneal the synthesized forward oligonucleotide sequence to the reverse oligonucleotide sequence, Form double-stranded oligonucleotides with cohesive ends;

(2) Link the above-mentioned double-stranded oligonucleotide into the expression vector lentiCRISPRv2 of the sequence shown in SEQIDNO: 39 in the sequence table, and obtain the linearized vector obtained by digesting with BsmBI restriction endonuclease to obtain the oligonucleotide carrying sgRNA The recombinant expression vector lentiCRISPRv2-GGTA1 of acid was transformed into competent bacteria, and the correct positive clones were screened and identified, and the positive clones were shaken and plasmids were extracted;

(3) Use the above-mentioned expression vector lentiCRISPRv2-GGTA1, packaging plasmid and packaging cell line to package a pseudotyped lentivirus carrying both sgRNA and Cas9 targeting the GGTA1 gene;

(4) Use the above-mentioned CRISPR pseudotyped lentivirus to infect the target cells, and further culture; then collect the infected target cells, use their genomic DNA as a template to amplify the gene fragment containing the above-mentioned target sequence, and undergo denaturation, renaturation and enzyme digestion , to determine the knockout status of the GGTA1 gene.

As a preferred solution of the present invention, the aforementioned packaging plasmids are plasmid pLP1, plasmid pLP2 and plasmid pLP/VSVG; the aforementioned packaging cell line is HEK293T cells.

As a preferred solution of the present invention, the above-mentioned target cells are porcine PIEC cells.

As a preferred solution of the present invention, the above-mentioned gene fragment containing the above-mentioned target sequence is amplified using its genomic DNA as a template, and after denaturation, renaturation and enzyme digestion, the knockout situation of the GGTA1 gene is determined, specifically:

(a) Using the genomic DNA of the target cell infected with the virus as a template, use the upstream and downstream primers of the GGTA1 gene to amplify the GGTA1 gene fragment containing the target sequence of the above sgRNA, and simultaneously use the same primers to amplify the genome of the wild-type cell that is not infected with the virus DNA;

(b) purifying the above-mentioned amplified GGTA1 gene fragment, then mixing the GGTA1 gene fragment from the target cell infected with the virus with the GGTA1 gene fragment from the wild-type cell in equal amounts, heat denaturation, and renaturation to form a hybrid DNA molecule;

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

(d) Electrophoresis detection of the enzyme cleavage product to detect the effect of the target sequence-mediated GGTA1 gene knockout.

According to the third aspect of the present invention, the present invention provides the recombinant expression vector lentiCRISPRv2-GGTA1 used in the method of CRISPR-Cas9 specific knockout of pig GGTA1 gene, the sequence of the backbone vector of the recombinant expression vector is shown in SEQ ID NO in the sequence listing: 39; the target sequence carried is as the target sequence of the sgRNA in the first aspect, preferably the target sequence shown in SEQ ID NO: 1 in the sequence listing.

According to the fourth aspect of the present invention, the present invention provides the use of the sgRNA described in the first aspect or the recombinant expression vector lentiCRISPRv2-GGTA1 described in the third aspect in a method for specifically knocking out the pig GGTA1 gene by CRISPR-Cas9.

The present invention aims at CRISPR-Cas9 specific knockout of the pig GGTA1 gene, and successfully finds the sgRNA specifically targeting the GGTA1 gene, and the sgRNA of the present invention is used in the method of CRISPR-Cas9 specific knockout of the pig GGTA1 gene, which can quickly The method can knock out the pig GGTA1 gene accurately, efficiently and specifically, and effectively solve the technical problems of long construction period and high cost of constructing GGTA1 gene knockout pigs.

Description of drawings

Fig. 1 is the plasmid map of the carrier plasmid lentiCRISPRv2 used in the embodiment of the present invention;

Fig. 2 is the plasmid map of the packaging plasmid pLP1 used in the embodiment of the present invention;

Fig. 3 is the plasmid map of the packaging plasmid pLP2 used in the embodiment of the present invention;

Fig. 4 is the plasmid map of the packaging plasmid pLP/VSVG used in the embodiment of the present invention;

Figure 5 is an electrophoretic detection result diagram of the gene knockout effect of the target sequence verified by enzyme digestion in the embodiment of the present invention, wherein M represents DNAMarker, Ctrl represents the targeted cutting effect of the control sequence that cannot effectively target the GGTA1 gene, and 1 represents Table 1 The No. 1 target sequence in Table 1 has the targeted cleavage effect on the GGTA1 gene, 3 represents the targeted cleavage effect of the No. 3 target sequence in Table 1 on the GGTA1 gene, and WT represents the PCR product of wild-type cells that have not been infected by the virus and cut by Cas9 Cruiser enzyme digestion results, the arrow indicates the small fragments obtained by Cruiser enzyme digestion;

Figure 6 is an electrophoresis detection result of the GGTA1 gene targeted cleavage effect of 6 PIEC cell monoclonals based on sequence 1 in the embodiment of the present invention. The arrows indicate the small fragments obtained by Cruiser enzyme cleavage. Except for weak cells, cells 1, 2, 3, 4, and 6 all had obvious cutting fragments;

Figure 7 is a diagram of the sequencing identification results of cells No. 1, 3 and 6 in Figure 6, showing that sequence 1 effectively produced the GGTA1 gene mutation, wherein the sequencing results of cell No. 1 (Fig. 7A) contained insertion mutations after analysis (Fig. 1 sequence in 7D); the sequencing result of No. 3 cell (Fig. 7B) contained deletion mutation after analysis (3 sequence in Fig. 7D); the sequencing result of No. 6 cell (Fig. 7C) contained deletion mutation after analysis (Fig. 6 sequence in 7D); the arrow in Figure 7A and B indicates the theoretical cleavage site of sequence 1 mediated by Cas9 on the GGTA1 gene, and WT indicates the wild-type sequence.

detailed description

The technical solutions of the present invention will be 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 present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

The test materials and reagents involved in the following examples: the lentiCRISPRv2 plasmid was purchased from Addgene, the packaging plasmids pLP1, pLP2 and pLP/VSVG were purchased from Invitrogen, and the packaging cell line HEK293T cells were purchased from the American Type Culture Collection (ATCC), PIEC cells were purchased from the Cell Bank of the Chinese Academy of Sciences, DMEM medium, Opti-MEM medium and fetal bovine serum FBS were purchased from Gibco, and Lipofectamine2000 was purchased from Invitrogen.

The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods described in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or carried out according to the kit and product instructions .

Generalized technical scheme of the present invention comprises following five parts:

1. Selection and design of Susscrofa (pig) GGTA1 gene sgRNA target sequence

1. Selection of sgRNA target sequence of GGTA1 gene:

Find a suitable 20bp oligonucleotide sequence in the exon region of GGTA1 gene as the target sequence.

2. sgRNA target sequence design of GGTA1 gene:

Linkers are added to the above target sequence and complementary sequence to form a forward oligonucleotide sequence and a reverse oligonucleotide sequence.

2. Construction of CRISPR vector for GGTA1 gene

1. Synthesize the above-mentioned forward oligonucleotide sequence and reverse oligonucleotide sequence, and anneal to form a double-stranded DNA fragment with sticky ends (that is, a double-stranded target sequence oligonucleotide, also known 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 (such as lentiCRISPRv2, whose plasmid map is shown in Figure 1), to form a lentiviral CRISPR vector such as lentiCRISPRv2-GGTA1.

3. Obtain a pseudotyped lentivirus expressing GGTA1sgRNA

CRISPR pseudotyped lentiviruses expressing GGTA1 sgRNA were produced using packaging plasmids, packaging cell lines and lentiviral CRISPR vectors.

4. Infect target cells and detect the knockout effect of GGTA1 gene

1. Infect target cells with lentivirus:

A pseudotyped lentivirus such as lentiCRISPRv2-GGTA1 is added to the target cell culture medium for infection and further culture.

2. Detection of GGTA1 gene knockout effect:

The target cells were collected, and the gene fragment containing the target sequence was amplified using genomic DNA as a template. After denaturation, renaturation and enzyme digestion, the knockout status of the GGTA1 gene was determined.

5. Selection and Identification of GGTA1 Gene Knockout Monoclonal

1. For the target cell population with a confirmed knockout effect, several cell lines derived from single cells are isolated by dilution and monoclonal culture.

2. Identification of monoclonal GGTA1 knockout.

The technical solutions of the present invention and their beneficial effects are described in detail below through examples.

Example 1, Selection and design of Susscrofa (pig) GGTA1 gene sgRNA target sequence

The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors.

1. Selection of sgRNA target sequence for GGTA1 gene

For the GGTA1 gene, the following principles should be followed in the selection of target sequences:

(1) Find the target sequence conforming to the 5'-N(20)NGG-3' rule in the exon coding region of the GGTA1 gene, where N(20) represents 20 consecutive bases, and each N represents A or T Or C or G, the target sequence that meets the above rules is located on the sense strand or the antisense strand;

(2) Select the 5 exon coding region sequences near the N-terminal, the target sequence can be located in the 5 exon coding regions of the N-terminal of the GGTA1 gene, or a part of the target sequence is located in the 5 exons of the N-terminal of the GGTA1 gene, The remaining part spans the junction with the adjacent intron and is located in the adjacent intron; the cleavage of such a coding region sequence will cause the functional knockout of the GGTA1 gene, and the remaining truncated sequence will not form a functional protein;

(3) If there are multiple splices, select in the common exon coding region, and select the 5 exon coding region sequences near the N-terminal of the GGTA1 gene to meet this condition;

(4) Use 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 score. The target sequence is unique on the GGTA1 gene.

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

Table 1 Target sequence set

Numbering sequence 1 GCTGCTTGTCTCAACTGTAA 2 TGTTTTGGGAATACATCAAC 3 CAACTGTAATGGTTGTGTTT 4 AACTGTAATGGTTGTGTTTT 5 TCCAGAACAAAGAACCTTCT 6 ATCCAGAACAAAGAACCTTC 7 TTCCGAGCTGGTTTAACAAT 8 TTTCCGAGCTGGTTTAACAA 9 GGGGCTGGTGGTTTCCGAGC 10 TGAGCACTGCTGCCAACTTC 11 GAGCACTGCTGCCAACTTCT

12 AAGTTGGCAGCAGTGCTCAG 13 GAGAGCTTCCGCTAGTGGAC 14 AGACGCTATAGGCAACGAAA 15 CAGAGGAGAGCTTCCGCTAG 16 GGATTAAACCAGTCCACTAG 17 CTATAGCGTCTTTCTTCTTCG 18 CACGAAGAAGAAGACGCTAT 19 TGGTTATGGTCACGACCTCT 20 CTGGTTATGGTCACGACCTC twenty one AGGTCGTGACCATAACCAGA twenty two TGACGGTTTTTTGCTGTCGGA twenty three GCCAAACAGAAAATTACCGT twenty four ATGGAAGGCTCCAGTGGTAT 25 TAAGTGCCTTCCCATACCAC 26 CACGACCTCTGGGCGTTTCC 27 GGTTTTTGCTGTCGGAAGGT 28 GCCCACGGTAATTTTCTGTT 29 GATGGAAGGCTCCAGTGGTA 30 AAGGCTCCAGTGGTATGGGA 31 TGCCAAACAGAAAATTACCG 32 GAAAATTACCGTGGGCTTGA 33 GGCTTGACGGTTTTTGCTGT 34 AGCAAAAAACCGTCAAAGCCCA 35 CGTGACCATAACCAGATGGA 36 TGGAGCCTTCCATCTGGTTA

37 GGCATAATAATTATCTAAGA 38 AACCAGATGGAAGGCTCCAG

2. sgRNA target sequence design of GGTA1 gene:

(1) Using the lentiCRISPRv2 plasmid as the expression vector, according to the characteristics of the lentiCRISPRv2 plasmid, a CACCG sequence was added to the 5'-end of the above N(20) target sequence to form a forward oligonucleotide sequence:

5'-CACCGNNNNNNNNNNNNNNNNNNNNN-3';

(2) Add sequences at both ends of the reverse complementary sequence of the above N(20) target sequence to form a reverse oligonucleotide sequence:

5'-AAACNNNNNNNNNNNNNNNNNNNNNC-3';

The forward and reverse oligonucleotide sequences can complement each other to form double-stranded DNA fragments with cohesive ends:

5'-CACCGNNNNNNNNNNNNNNNNNNNNN-3'

3'- CNNNNNNNNNNNNNNNNNNNNCAAA-5'.

Example 2: Construction of the sgRNA expression vector of the GGTA1 gene

1. Synthesis of DNA Inserts

(1) Synthesize the forward and reverse oligonucleotide sequences designed above

The oligonucleotide sequence can be specifically synthesized by a commercial company (such as Invitrogen) according to the provided sequence. In this example and the following examples, the knockout effect of the target sequence shown in the No. 1 sequence listed in Table 1 on the GGTA1 gene was studied.

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

CACCGGCTGCTTGTCTCAACTGTAA (SEQ ID NO: 40);

AAACTTACAGTTGAGACAAGCAGCC (SEQ ID NO: 41).

The corresponding forward and reverse oligonucleotide sequences are annealed and annealed to form double-stranded DNA fragments with cohesive ends.

The reaction system (20μL) is as follows:

Forward oligonucleotide (10 μM): 1 μL

Reverse oligonucleotide (10 μM): 1 μL

10×PCR buffer: 2μL

_ddHO : 16 μL

Put the above reaction system into the PCR machine, and carry out the reaction according to the following procedure.

Reaction procedure:

95℃, 5min;

80℃, 5min;

70℃, 5min;

60℃, 5min;

50℃, 5min;

Cool down to room temperature naturally.

2. Construction of sgRNA expression vector

(1) Digest the target vector lentiCRISPRv2 plasmid (its sequence is shown in SEQ ID NO: 39 in the sequence listing) with BsmBI restriction endonuclease.

Prepare according to the following reaction system:

LentiCRISPRv2 plasmid: 1 μg

10×digestion buffer: 2μL

BsmBI restriction enzyme: 2 μL

Supplement _ddHO to a total volume of 20 μL

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

(2) Separation and purification of carrier fragments by electrophoresis

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

(3) Ligate the synthesized double-stranded DNA fragment with the vector main fragment and transform Escherichia coli

Ligate the double-stranded DNA fragments obtained by renaturation and the recovered carrier fragments, and prepare according to the following reaction system:

LentiCRISPRv2 vector fragment: 100ng

Double-stranded DNA fragment: 200ng

T4 ligase: 1 μL

T4 ligation reaction buffer: 1 μL

Supplement _ddHO to a total volume of 10 μL

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

After the reaction, the ligation mixture was transformed into Escherichia coli DH5α strain: add 100 μL of Escherichia coli DH5α competent cells to the ligation mixture, and incubate on ice for 30 min; put the mixture in a water bath at 42°C, heat shock for 90 s and cool it on ice; Add 100 μL LB medium, and incubate on a shaker at 37°C for 20 minutes; spread the mixture on an AmpLB plate, and incubate at 37°C for 14h.

(4) Identification of correct transformed clones

A number of colonies were selected from the AmpLB plate for expansion culture, and plasmids were extracted for enzyme digestion identification. Potentially correct clones were picked for sequencing to verify that the insert sequence was correct. Preserve correct lentiCRISPRv2-GGTA1 vector clones.

Example 3. Obtaining a pseudotyped lentivirus expressing GGTA1 sgRNA

1. Material preparation

Amplify and extract packaging plasmids pLP1, pLP2, and pLP/VSVG (purchased from Invitrogen, whose maps are shown in Figure 2, Figure 3, and Figure 4, respectively); amplify and extract vector plasmid lentiCRISPRv2-GGTA1; cultivate packaging cell lines HEK293T cells (purchased from ATCC); DMEM medium, Opti-MEM medium and fetal bovine serum FBS (purchased from Gibco); Lipofectamine2000 (purchased from Invitrogen); HEK293T cells were cultured in a 37°C culture environment containing 5% CO ₂ , the medium is DMEM medium containing 10% FBS.

2. Transfection and Viral Packaging

Day 1: Passage the packaging cell line HEK293T to 10cmdish, about 30% confluence;

The next day: when HEK293T reaches 80% confluence, transfect according to the following recipe:

Prepare Mixture 1, containing:

lentiCRISPRv2-GGTA1: 6 μg

pLP1: 6 μg

pLP2: 6 μg

pLP/VSVG: 3 μg

Opti-MEM: 500 μL.

Prepare Mixture 2, containing:

Lipofectamine2000: 30 μL

Opti-MEM: 500 μL.

After standing still for 5 minutes, mix mixture 1 and mixture 2 to form a transfection mixture, and let stand for 20 minutes.

The HEK293T medium was replaced with a serum-free DMEM medium, and the transfection mixture was added, cultured at 37° C. for 8 hours, then replaced with 20% FBS DMEM medium, and the culture continued.

3. Virus collection and preservation

Day 3: 48 hours after transfection, the HEK293T medium supernatant containing the virus was collected, filtered with a 0.45 μm filter head, aliquoted, and stored at -80°C.

Example 4. Infecting Target Cells and Detecting the Knockout Effect of the Target Sequence

1. Material preparation

Culture the target cell line porcine hip artery vascular endothelial cells PIEC (purchased from the Cell Bank of the Chinese Academy of Sciences); DMEM medium and fetal bovine serum FBS (purchased from Gibco); lentiCRISPRv2-GGTA1 pseudotypes of different target sequences (sequence 1 and control sequence) Lentivirus; PIEC cells were cultured in a 37°C culture environment containing 5% CO ₂ , and the medium was DMEM medium containing 10% FBS.

2. Infect target cells with lentivirus

Day 1: Passage the target cells to a 6-well plate at about 20% confluent density. Each virus needs a 6-well, and an efficiency control needs a 6-well.

The next day: Add 1mL lentiCRISPRv2-GGTA1 pseudotyped lentiviral supernatant and 1mL DMEM medium when the target cells are about 40% confluent. The efficiency control does not require the addition of lentivirus.

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

3. Cell infection efficiency detection and culture

Day 5: The uninfected efficiency control cells should all be apoptotic (>95%) under the action of puromycin. Judging the infection efficiency of the cells according to the apoptosis of the infected lentivirus cells, usually an infection efficiency of more than 90% can be achieved (apoptosis rate <10%). If necessary, the virus supernatant can be concentrated or serially diluted before infection to achieve a suitable infection efficiency.

After puromycin selection, uninfected cells undergo apoptosis. The target cells were subcultured again and replaced with normal medium for 48 h.

4. Detection of GGTA1 gene knockout effect

(1) Design upstream and downstream primers to amplify the GGTA1 gene fragment, wherein the sequences of the upstream and downstream primers are as follows:

ctgtcagttcattgacttggctaatttgc (SEQ ID NO: 42)

caagctggtgacttggctgataactag (SEQ ID NO: 43).

The target amplified fragment contains the sgRNA target sequence and is 362bp in size. The position from the target sequence to both ends of the fragment is not less than 100bp.

(2) Collect some target cells, and use promega genomic DNA kit to extract genomic DNA. At the same time, the genomic DNA of the wild-type target cells was extracted.

(3) Using genomic DNA as a template to amplify the GGTA1 gene fragment containing the target sequence (including infected mutant samples and wild-type samples).

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

Upstream primer (10 μM): 1 μL

Downstream primer (10μM): 1μL

2 × PCR Mix: 10 μL

Genomic DNA: 100ng

Prepare the above reaction system, put it into the PCR machine, and carry out the reaction according to the following procedures.

Reaction procedure:

95℃,3min

95℃,30s

58℃,20s

72℃,20s

72℃, 3min;

Wherein the second step to the fourth step repeat 35 cycles.

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

(5) Heat the purified DNA fragments to denature and renature, respectively, 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: 2μL

Total reaction system 9μL

Prepare the above reaction system, put it into the PCR machine, and carry out the reaction according to the following procedures.

Reaction procedure:

95℃, 5min;

80℃, 5min;

70℃, 5min;

60℃, 5min;

50℃, 5min;

Cool down to room temperature naturally.

(6) Cutting the renatured hybrid DNA (including mutant samples and wild-type samples) with Cruiser enzyme Add 1 μL Cruiser enzyme to the denatured and renatured reaction mixture, and incubate at 45° C. for 20 min.

(7) Electrophoresis to detect the digested product, and to detect the effect of the target sequence-mediated GGTA1 gene knockout.

The digested DNA fragments were analyzed by electrophoresis on 2% agarose gel, 100V, 25min. Determine the cleavage of the target fragment and judge the gene knockout effect of the target sequence.

Cleavage recognition of mutant DNA is based on the principle that infected cells express sgRNA and Cas9. If the genomic DNA is cleaved by the sgRNA-mediated Cas9 protein, mutations will be introduced near the cleavage site after repair (wild type becomes mutant). Since the wild-type and mutant-type sequences do not match at this position, the hybrid molecule formed by denaturing the wild-type DNA and the mutant-type DNA amplified using this as a template will produce a local loop (loop) structure. The latter can be recognized and cut by Cruiser enzyme, resulting in the cutting of hybrid DNA molecules into small fragments.

As a result, as shown in Figure 5, the control sequence (SEQIDNO:44GAATACATCAACAGCCCAGA in the sequence listing) cannot effectively target the GGTA1 gene to produce cleavage, so small fragments are not detected; Small fragments were detected; no obvious cleavage band was detected for sequence 3, which may be due to the ineffective targeting of the target genomic DNA, or the low efficiency of targeted cleavage is difficult to observe. Another possible reason is that if the PIEC cells used in the examples have a polymorphism at this site, which is inconsistent with the standard sequence, sequence 3 will not be effectively targeted. Sequence 1 can effectively target the GGTA1 gene to produce cleavage, so the existence of small fragments was detected, indicating that Sequence 1 can be used as a target sequence for CRISPR-Cas9 to specifically knock out the pig GGTA1 gene.

Example 5, Selection and Identification of GGTA1 Gene Knockout Single Clones

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

(1) Part of the infected target cell population was subcultured, and 100 single cells were transferred to 10cmdish for culture.

(2) After culturing for about 10 days, a considerable number of monoclonal growths reached the level visible to the naked eye.

(3) Use a pipette tip to scrape independent clones, transfer the cells to a 24-well plate for culture, and each well corresponds to a clone.

(4) After about one week of cultivation, some clones grow to a sufficient number and are ready for further identification.

2. Identification of monoclonal GGTA1 knockout

(1) Collect monoclonal and wild-type cells to be tested, and extract genomic DNA respectively.

(2) According to the aforementioned method, the GGTA1 gene fragments of the monoclonal and wild-type cells were respectively amplified, and the amplified gene fragments contained the sgRNA target sequence.

(3) Mix equal amounts of monoclonal PCR fragments and wild-type PCR fragments, heat denature and renature to form hybrid DNA molecules.

(4) Cut the annealed hybridized DNA with Cruiser enzyme and incubate at 45°C for 20min.

(5) Electrophoresis to detect the digested product, and determine whether the monoclonal has an effective mutation according to whether there is a cleaved fragment ( FIG. 6 ).

(6) Further sequence the PCR fragments of the effectively mutated monoclonals to determine the mutations near the target sequence. A single clone knocked out of the GGTA1 gene was identified (Fig. 7).

The results shown in Figure 6 show that the lentiCRISPRv2-GGTA1 pseudotyped lentivirus based on the target sequence shown in Sequence 1 infected the target cells, and 6 single clones randomly selected from 100 single cells were detected by Cruiser enzyme digestion electrophoresis, among which All 6 monoclonals (No. 5 monoclonal band is weaker) can detect small cleavage fragments, indicating that gene knockout occurs, and the gene knockout efficiency can reach 100%, indicating that the target sequence shown in sequence 1 has a high Effect of targeted knockout of the GGTA1 gene.

Figure 7 shows the further sequencing of the PCR fragments of No. 1, No. 3 and No. 6 single-cell clones in Fig. 6 to determine the mutation situation near the cutting site, wherein Fig. 7A is the sequencing result of the PCR fragment of No. 1 single-cell clone , the analysis contained an insertion mutation (sequence 1 in Figure 7D, with a G nucleotide inserted). This indicates that a frameshift mutation occurred, which can clearly illustrate the successful targeted knockdown of the GGTA1 gene. Compared with the wild-type sequence, it was found that the inserted G was located at the theoretical cleavage site.

Fig. 7B is the sequencing result of the PCR fragment of No. 3 single-cell clone, which contains a deletion mutation (sequence 3 in Fig. 7D ) after analysis. Figure 7C is the sequencing result of the PCR fragment of single cell clone No. 6, which contains another deletion mutation (sequence 6 in Figure 7D) after analysis. The theoretical cleavage site of No. 6 single-cell clone has disappeared after being repaired. Both monoclonal sequences showed a frameshift, indicating a targeted knockout of the GGTA1 gene. The presence of the above insertion mutations and deletion mutations indicates that Sequence 1 can efficiently mediate targeted knockout of the GGTA1 gene.

The above content is a further detailed description of the present invention in conjunction with specific embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. Those of ordinary skill in the technical field to which the present invention belongs can also make some simple deduction or replacement without departing from the concept of the present invention.

Claims (10)

1. using the sgRNA for selectively targeted GGTA1 gene in CRISPR-Cas9 specific knockdown pig GGTA1 gene, it is characterized in that:

(1) target sequence of described sgRNA on GGTA1 gene meets the series arrangement rule of 5 '-N (20) NGG-3 ', wherein N (20) represents 20 continuous print bases, wherein each N represents A or T or C or G, and the target sequence meeting described rule is positioned at positive-sense strand or antisense strand;

(2) target sequence of described sgRNA on GGTA1 gene is positioned at 5 exons coding districts of the N end of GGTA1 gene, or a part for target sequence is positioned at 5 exons of the N end of GGTA1 gene, rest part crosses over the boundary with adjacent intron, is positioned at adjacent intron;

(3) target sequence of described sgRNA on GGTA1 gene is unique.

2. the sgRNA for selectively targeted GGTA1 gene according to claim 1, is characterized in that, described target sequence is the sequence shown in arbitrary sequence in SEQ ID NO:1 ~ 38.

3. the sgRNA for selectively targeted GGTA1 gene according to claim 1, is characterized in that, described target sequence is the sequence shown in SEQ ID NO:1.

4. use the method for CRISPR-Cas9 specific knockdown pig GGTA1 gene, it is characterized in that, described method comprises the steps:

(1) 5 '-end of the target sequence of the sgRNA described in any one of claim 1-3 adds the sequence for the formation of sticky end, and synthesis obtains forward oligonucleotide sequence; The two ends of the complementary sequence that the target sequence of the sgRNA described in any one of claim 1-3 is corresponding add the suitable sequence for the formation of sticky end, and synthesis obtains reverse oligonucleotide sequence; By the described forward oligonucleotide sequence of synthesis and reverse oligonucleotide sequence anneals, renaturation, form the double stranded oligonucleotide with sticky end;

(2) described double stranded oligonucleotide is connected into the linearizing expression vector carrying Cas9 gene, obtain carrying the expression vector of sgRNA oligonucleotide containing respective target sequence and Cas9 gene, transform competent bacteria, Screening and Identification goes out correct positive colony, and described positive colony is shaken to bacterium, extracts plasmid;

(3) the false type slow virus that the expression vector of sgRNA oligonucleotide and Cas9 gene, packaging plasmid and package cell line pack out sgRNA and Cas9 simultaneously carrying target GGTA1 gene is carried described in use;

(4) use described false type slow virus infection object cell, and cultivate further; Then collect infected object cell, with the gene fragment of its genomic dna described target sequence for template amplification comprises, cut through sex change, renaturation and enzyme, that determines GGTA1 gene knocks out situation.

5. the method for CRISPR-Cas9 specific knockdown pig GGTA1 gene according to claim 4, it is characterized in that, described expression vector is the carrier of sequence shown in SEQ ID NO:39.

6. the method for the CRISPR-Cas9 specific knockdown pig GGTA1 gene according to claim 4 or 5, it is characterized in that, described method comprises the steps:

(1) 5 '-end of the target sequence of the sgRNA described in any one of claim 1-3 adds CACCG sequence, and synthesis obtains forward oligonucleotide sequence; 5 '-end of the complementary sequence that the target sequence of the sgRNA described in any one of claim 1-3 is corresponding adds AAAC sequence, 3 '-end adds C, and synthesis obtains reverse oligonucleotide sequence; By the described forward oligonucleotide sequence of synthesis and reverse oligonucleotide sequence anneals, renaturation, form the double stranded oligonucleotide with sticky end;

(2) linearized vector that the expression vector lentiCRISPRv2 described double stranded oligonucleotide being connected into sequence as shown in SEQ ID NO:39 obtains through BsmBI digestion with restriction enzyme, obtain the recombinant expression vector lentiCRISPRv2-GGTA1 carrying sgRNA oligonucleotide, transform competent bacteria, Screening and Identification goes out correct positive colony, and described positive colony is shaken to bacterium, extracts plasmid;

(3) the false type slow virus of sgRNA and Cas9 simultaneously carrying target GGTA1 gene is packed out with described expression vector lentiCRISPRv2-GGTA1, packaging plasmid and package cell line;

(4) use described false type slow virus infection object cell, and cultivate further; Then collect infected object cell, with the gene fragment of its genomic dna described target sequence for template amplification comprises, cut through sex change, renaturation and enzyme, that determines GGTA1 gene knocks out situation.

7. the method for CRISPR-Cas9 specific knockdown pig GGTA1 gene according to claim 6, it is characterized in that, described packaging plasmid is plasmid pLP1, plasmid pLP2 and plasmid pLP/VSVG; Described packing cell is HEK293T cell.

8. the method for CRISPR-Cas9 specific knockdown pig GGTA1 gene according to claim 6, it is characterized in that, described object cell is pig PIEC cell;

Described with the gene fragment of its genomic dna described target sequence for template amplification comprises, cut through sex change, renaturation and enzyme, that determines GGTA1 gene knocks out situation, is specially:

A () is to infect the genomic dna of the object cell of virus for template, the GGTA1 gene fragment of the target sequence of described sgRNA is comprised, simultaneously with the genomic dna of the wild-type cell of same primers amplification uninfecting virus with the upstream and downstream primer amplification of GGTA1 gene;

B GGTA1 gene fragment that the above-mentioned amplification of () purifying is arrived, then in the future the GGTA1 gene fragment of the object cell of self-infection virus and GGTA1 gene fragment balanced mix, heat denatured, the renaturation from wild-type cell, form hybrid DNA molecule;

C () cuts the hybrid DNA molecule after renaturation with Cruiser enzyme;

D () electrophoresis detection digestion products, detects the GGTA1 gene knockout effect of target sequence mediation.

9. the recombinant expression vector lentiCRISPRv2-GGTA1 used in the method for CRISPR-Cas9 specific knockdown pig GGTA1 gene, it is characterized in that, the sequence of the skeleton carrier of described recombinant expression vector is as shown in SEQ ID NO:39; The target sequence of the entrained sgRNA of target sequence as described in any one of claim 1-3, the target sequence shown in SEQIDNO:1 in preferred sequence table.

10. the purposes of the sgRNA as described in any one of claim 1-3 or recombinant expression vector lentiCRISPRv2-GGTA1 according to claim 9 in the method for CRISPR-Cas9 specific knockdown pig GGTA1 gene.