CN106414740A - CRISPR-Cas9 method for specifically knocking out porcine SLA-3 gene and sgRNA for specifically targeting SLA-3 gene - Google Patents

Abstract

A method for specifically knocking out pig SLA-3 gene by using CRISPR-CaS9 and sgRNA for specifically targeting SLA-3 gene are provided. The target sequence of the sgRNA specifically targeting the SLA-3 gene on the SLA-3 gene is positioned at the junction of 5 exon coding regions at the N end of the SLA-3 gene and adjacent introns.

Description

CRISPR-Cas9 method for specifically knocking out pig SLA-3 gene and its use for specific target sgRNA to SLA-3 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 SLA-3 gene by CRISPR-Cas9 and an sgRNA for specifically targeting the SLA-3 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, genetically engineering pigs to produce organs suitable for human transplantation has become the ultimate goal of xenotransplantation.

Swine leukocyte antigen (swine leukocyte antigen, SLA) class I molecules are important functional genes that represent the molecular genetic characteristics of genes. They are called SLA-1 (PD1), SLA-2 (PD14), and SLA-3 (PD7), respectively, and are also called SLA-C, SLA-B, and SLA-A, respectively. The classic SIJA gene is mainly highly expressed in the immune system and digestive system, especially in the immune system, the spleen, thymus, bronchial lymph nodes and other tissues are rich in immune cells. Human CD4 T cells recognize porcine xenoantigens through an indirect antigen presentation pathway, and are mainly recognized by SLA class I molecules. Therefore, there is a need to eliminate SLA class I molecules on the surface of porcine cells to reduce the immunogenicity of xenogeneic donor organs. The accurate and efficient knockout of pig SLA-3 gene is a key step to eliminate the immune rejection caused by SLA class I molecules.

At present, common gene knockout technologies include Homologus Recombination (Homologus Recombination, HR) technology, Transcription Activator-Like Effector Nuclease (TALEN) technology, Zinc-Finger Nuclease (ZFN ) technology and the recently developed technology of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). 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 (CRISPR RNA) and tracrRNA (trans-activating crRNA)—to the target sequence. Now two small RNAs have been fused into one RNA chain, referred to as sgRNA (single guide RNA), 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 SLA-3 gene by CRISPR technology can greatly improve the screening efficiency of Neu5Gc-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 SLA-3 gene.

Contents of the invention

The purpose of the present invention is to provide a CRISPR-Cas9 method for specifically knocking out the pig SLA-3 gene and an sgRNA for specifically targeting the SLA-3 gene.

According to the first aspect of the present invention, the present invention provides an sgRNA for specifically targeting the SLA-3 gene in CRISPR-Cas9 specific knockout of the pig SLA-3 gene, the sgRNA has the following characteristics:

(1) The target sequence of the sgRNA on the SLA-3 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 conforming to the rules can be located on the sense strand or the antisense strand;

(2) The target sequence of the sgRNA on the SLA-3 gene is located in the 5 exons coding region of the N-terminus of the SLA-3 gene, or the main part of the sequence is located in the 5 exons of the N-terminus of the SLA-3 gene, and the rest Partially spanning the junction with the adjacent intron, located in the adjacent intron;

(3) The target sequence of the sgRNA on the SLA-3 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: 1-115 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: 4, 5 or 12 in the sequence listing.

According to a second aspect of the present invention, the present invention provides a method for CRISPR-Cas9 specific knockout of the pig SLA-3 gene, the method comprising the steps of:

(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, and synthesize the reverse oligonucleotide sequence; anneal the synthesized forward oligonucleotide sequence and reverse oligonucleotide sequence, 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) Packaging a pseudotyped lentivirus carrying both sgRNA and Cas9 targeting the SLA-3 gene with the above-mentioned expression vector, packaging plasmid and packaging cell line carrying the sgRNA oligonucleotide and the Cas9 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 SLA-3 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: 116 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 lentiCRISPR v2 of the sequence shown in SEQ ID NO: 116 in the sequence listing, and obtain the linearized vector carrying sgRNA The oligonucleotide recombinant expression vector lentiCRISPR v2-SLA-3 was transformed into competent bacteria, the correct positive clones were screened and identified, and the positive clones were shaken to extract plasmids;

(3) Use the above-mentioned expression vector lentiCRISPR v2-SLA-3, packaging plasmid and packaging cell line to package a pseudotyped lentivirus carrying both sgRNA and Cas9 targeting the SLA-3 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 SLA-3 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 genomic DNA is used as a template to amplify the gene fragment containing the above-mentioned target sequence, and after denaturation, renaturation and enzyme digestion, the knockout situation of the SLA-3 gene is determined, specifically:

(a) Using the genomic DNA of the virus-infected target cell as a template, use the upstream and downstream primers of the SLA-3 gene to amplify the SLA-3 gene fragment containing the target sequence of the above sgRNA, and simultaneously use the same primers to amplify the wild-type cells that are not infected with the virus Genomic DNA of type cells;

(b) Purify the above-mentioned amplified SLA-3 gene fragment, then mix the SLA-3 gene fragment from the virus-infected target cell with the SLA-3 gene fragment from the wild-type cell in equal amounts, heat denaturation and renaturation, Formation of hybrid DNA molecules;

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

(d) Electrophoresis to detect the digested product, and detect the target sequence-mediated SLA-3 gene knockout effect.

According to the third aspect of the present invention, the present invention provides the recombinant expression vector lentiCRISPR v2-SLA-3 used in the method of CRISPR-Cas9 specific knockout of pig SLA-3 gene, the sequence of the backbone vector of the recombinant expression vector is as follows: Shown in SEQ ID NO: 116 in the sequence listing; 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: 4, 5 or 12 in the sequence listing.

According to the fourth aspect of the present invention, the present invention provides a method for specifically knocking out the pig SLA-3 gene in CRISPR-Cas9 using the sgRNA described in the first aspect or the recombinant expression vector lentiCRISPR v2-SLA-3 described in the third aspect use in .

The invention specifically knocks out the pig SLA-3 gene by CRISPR-Cas9, successfully finds the sgRNA specifically targeting the SLA-3 gene, and uses the sgRNA of the invention to specifically knock out the pig SLA-3 gene by CRISPR-Cas9 In the method, the pig SLA-3 gene can be knocked out quickly, accurately, efficiently and specifically, and the technical problems of long construction period and high cost of constructing the SLA-3 gene knockout pig can be effectively solved.

Description of drawings

Fig. 1 is the plasmid map of the carrier plasmid lentiCRISPR v2 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 indicates DNA Marker, and 4, 5 and 12 respectively indicate No. 4, No. 5 and No. 12 in Table 1 Target sequence No. cleavage effect on SLA-3 gene, WT represents the PCR product Cruiser digestion detection results of wild-type cells that have not been infected by virus and Cas9 cleavage, and the arrows represent small fragments cleaved by Cruiser enzyme.

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: 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 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 Lipofectamine 2000 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 Sus scrofa (pig) SLA-3 gene sgRNA target sequence

1. SgRNA target sequence selection of SLA-3 gene:

Find a suitable 20bp oligonucleotide sequence in the exon region of SLA-3 gene as the target sequence.

2. Design of sgRNA target sequence of SLA-3 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 SLA-3 gene

1. Synthesize the above-mentioned forward oligonucleotide sequence and reverse oligonucleotide sequence, and anneal to form a double-stranded DNA fragment (ie, double-stranded target sequence oligonucleotide) with cohesive ends.

2. Construction of CRISPR-sgRNA expression vector:

The above double-stranded DNA fragments were constructed into a target vector (such as lentiCRISPR v2, whose plasmid map is shown in Figure 1), to form a lentiviral CRISPR vector such as lentiCRISPR v2-SLA-3.

3. Obtain a pseudotyped lentivirus expressing SLA-3sgRNA

CRISPR pseudotyped lentiviruses expressing SLA-3 sgRNA were produced using packaging plasmids, packaging cell lines and lentiviral CRISPR vectors.

4. Infect target cells and detect the knockout effect of SLA-3 gene

1. Infect target cells with lentivirus:

A pseudotyped lentivirus such as lentiCRISPR v2-SLA-3 is added to the target cell culture medium for infection and further culture.

2. Detection of SLA-3 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 SLA-3 gene was determined.

5. Selection and Identification of SLA-3 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. Identify the knockout of SLA-3 in monoclonal.

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

Example 1, Selection and design of Sus scrofa (pig) SLA-3 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. SgRNA target sequence selection of SLA-3 gene

For the SLA-3 gene, the following principles should be followed in the selection of target sequences:

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

(2) Select the 5 exon coding region sequences near the N-terminal, the cutting of such coding region sequences will cause the functional knockout of the SLA-3 gene, and the remaining truncated sequences will not form functional proteins;

(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 SLA-3 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 SLA-3 gene.

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

Table 1 Target sequence set

2. Design of sgRNA target sequence of SLA-3 gene:

(1) Using the lentiCRISPR v2 plasmid as the expression vector, according to the characteristics of the lentiCRISPR v2 plasmid, add a CACCG sequence 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'.

Embodiment 2, constructing the sgRNA expression vector of SLA-3 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 effect of the target sequences listed in the No. 4, No. 5 and No. 12 sequences listed in Table 1 on the knockout effect of the SLA-3 gene was studied.

The forward oligonucleotide sequence and reverse oligonucleotide sequence corresponding to No. 4 target sequence are as follows:

5'-CACCGGGCCCTGACTGGTACCCGGG-3' (SEQ ID NO: 117);

5'-AAACCCCGGGTACCAGTCAGGGCCC-3' (SEQ ID NO: 118).

The forward oligonucleotide sequence and reverse oligonucleotide sequence corresponding to No. 5 target sequence are as follows:

5'-CACCGGCCCTGACTGGTACCCGGGA-3' (SEQ ID NO: 119);

5'-AAACTCCCGGGTACCAGTCAGGGCC-3' (SEQ ID NO: 120).

The forward oligonucleotide sequence and reverse oligonucleotide sequence corresponding to No. 12 target sequence are as follows:

5'-CACCGCCTGGCCCTGACTGGTACCC-3' (SEQ ID NO: 121);

5'-AAACGGGTACCAGTCAGGGCCAGGC-3' (SEQ ID NO: 122).

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 lentiCRISPR v2 plasmid (its sequence is shown in SEQ ID NO: 116 in the sequence listing) with BsmB I restriction endonuclease.

Prepare according to the following reaction system:

LentiCRISPR v2 plasmid: 1 μg

10×digestion buffer: 2μL

BsmB I 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:

LentiCRISPR v2 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, transform the ligation mixture 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 42°C water bath, heat shock for 90 seconds, and cool it on ice; Add 100 μL of LB medium and incubate on a shaker at 37°C for 20 minutes; spread the mixture on an Amp LB plate and incubate at 37°C for 14 hours.

(4) Identification of correct transformed clones

A number of colonies were selected from the Amp LB 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 lentiCRISPR v2-SLA-3 vector clones.

Example 3. Obtaining a pseudotyped lentivirus expressing SLA-3sgRNA

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 lentiCRISPR v2-SLA-3; culture Packaging cell line 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 at 37°C with 5% CO ₂ In the culture environment, the medium is DMEM medium containing 10% FBS.

2. Transfection and Viral Packaging

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

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

Prepare Mixture 1, containing:

lentiCRISPR v2-SLA-3: 6 μg

pLP1: 6 μg

pLP2: 6 μg

pLP/VSVG: 3 μg

Opti-MEM: 500 μL.

Prepare Mixture 2, containing:

Lipofectamine 2000: 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 storage

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); lentiCRISPR v2 of different target sequences (sequence 4, sequence 5 and sequence 12) -SLA-3 pseudotyped 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 lentiCRISPR v2-SLA-3 pseudotyped lentivirus supernatant and 1mL DMEM medium when the target cells are about 40% confluent. Efficiency controls do not require the addition of lentivirus.

Day 3: After 24 hours of infection, the virus-containing medium was removed and replaced with a 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 SLA-3 gene knockout effect

(1) Design upstream and downstream primers to amplify the SLA-3 gene fragment, wherein the upstream and downstream primer sequences are as follows:

CTCGGGGTCTCAGGCTCCAGGGCGG (SEQ ID NO: 123)

GGGCAGGAAACAAGGGAGGG (SEQ ID NO: 124).

The target amplified fragment contains the sgRNA target sequence with a size of 487bp. 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 SLA-3 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) Cut the renatured hybrid DNA with Cruiser enzyme (including mutant samples and wild-type samples)

Add 1 μL of Cruiser enzyme to the denatured and refolded reaction mixture, and incubate at 45°C for 20 minutes.

(7) Electrophoresis detection of enzyme cleavage products to detect the effect of target sequence-mediated SLA-3 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.

The results are shown in Figure 5, no small fragments were detected in the PCR products of wild-type cells that had not been infected with the virus; while Sequence 4, Sequence 5 and Sequence 12 could effectively target the SLA-3 gene to produce cleavage, so small fragments were detected Existence indicates that Sequence 4, Sequence 5 and Sequence 12 can be used as target sequences for CRISPR-Cas9 to specifically knock out the pig SLA-3 gene.

Example 5, Selection and Identification of SLA-3 Gene Knockout Monoclonal

1. Selection of monoclonal (target sequence based on sequence 4, sequence 5 and sequence 12)

(1) Part of the infected target cell population was subcultured, and 100 single cells were transferred to a 10cm dish 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 SLA-3 knockout

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

(2) According to the aforementioned method, the SLA-3 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 there is an effective mutation in the monoclonal according to whether there is a cleaved fragment.

The results showed that the lentiCRISPR v2-SLA-3 pseudotyped lentivirus based on the target sequence shown in sequence 4 infected the target cells, and 20 single clones randomly selected from 100 single cells were detected by Cruiser enzyme digestion electrophoresis, of which 17 A single clone can detect small cleavage fragments, indicating that gene knockout occurs, and the gene knockout efficiency can reach more than 85%, indicating that the target sequence shown in sequence 4 has a high targeted knockout effect on the SLA-3 gene. The lentiCRISPR v2-SLA-3 pseudotyped lentivirus based on the target sequence shown in Sequence 5 infected the target cells, and 20 single clones randomly selected from 100 single cells were detected by Cruiser enzyme digestion electrophoresis, and 19 single clones were detected Small cleavage fragments can be detected, indicating that gene knockout occurs, and the gene knockout efficiency can reach more than 95%, indicating that the target sequence shown in sequence 5 has a high targeted knockout effect on the SLA-3 gene. The lentiCRISPR v2-SLA-3 pseudotyped lentivirus based on the target sequence shown in Sequence 12 infected target cells, and 20 single clones randomly selected from 100 single cells were detected by Cruiser enzyme digestion electrophoresis, of which 18 single clones Small cleavage fragments can be detected, indicating that gene knockout occurs, and the gene knockout efficiency can reach more than 90%, indicating that the target sequence shown in sequence 12 has a high targeted knockout effect on the SLA-3 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. The sgRNA for specifically targeting the SLA-3 gene in the CRISPR-Cas9 specific knockout pig SLA-3 gene is characterized in that: (1) The target sequence of the sgRNA on the SLA-3 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 conforming to the rules can be located on the sense strand or the antisense strand; (2) The target sequence of the sgRNA on the SLA-3 gene is located in the five exon coding regions of the N-terminus of the SLA-3 gene, or the main part of the sequence is located in the five exons of the N-terminus of the SLA-3 gene, The remainder spans the junction with the adjacent intron and is located in the adjacent intron; (3) The target sequence of the sgRNA on the SLA-3 gene is unique. 2. The sgRNA for specifically targeting the SLA-3 gene according to claim 1, wherein the target sequence is a sequence shown in any one of SEQ ID NO: 1 to 115 in the sequence listing. 3. The sgRNA for specifically targeting the SLA-3 gene according to claim 1, wherein the target sequence is the sequence shown in SEQ ID NO: 4, 5 or 12 in the sequence listing. 4. The method for CRISPR-Cas9 specific knockout pig SLA-3 gene, is characterized in that, described method comprises the steps: (1) Add a sequence for forming a cohesive end to the 5'-end of the sgRNA target sequence according to any one of claims 1-3, and synthesize a forward oligonucleotide sequence; in claims 1-3 The two ends of the complementary sequence corresponding to the target sequence of the sgRNA described in any one add a suitable sequence for forming a cohesive end, and synthesize a reverse oligonucleotide sequence; the synthesized forward oligonucleotide The sequence is annealed and annealed with the reverse oligonucleotide sequence to form a double-stranded oligonucleotide with sticky ends; (2) connecting the double-stranded oligonucleotide into the 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 competent bacteria, Screening and identifying the correct positive clones, shaking the positive clones and extracting plasmids; (3) using the expression vector, packaging plasmid and packaging cell line carrying the sgRNA oligonucleotide and the Cas9 gene to package a pseudotyped lentivirus carrying both the sgRNA targeting the SLA-3 gene and the Cas9; (4) Use the pseudotyped lentivirus to infect the target cells and further cultivate them; then collect the infected target cells, use their genomic DNA as a template to amplify the gene fragment containing the target sequence, and undergo denaturation, renaturation and enzymatic Cut to determine the knockout status of the SLA-3 gene. 5. The method for CRISPR-Cas9 specific knockout of pig SLA-3 gene according to claim 4, characterized in that, the expression vector is the carrier of the sequence shown in SEQ ID NO: 116 in the sequence listing. 6. according to the method for the CRISPR-Cas9 specific knockout pig SLA-3 gene described in claim 4 or 5, it is characterized in that, described method comprises the steps: (1) Add a CACCG sequence to the 5'-end of the sgRNA target sequence according to any one of claims 1-3, and synthesize a forward oligonucleotide sequence; any one of claims 1-3 The 5'-end of the complementary sequence corresponding to the target sequence of the sgRNA is added with the AAAC sequence, and the 3'-end is added with C, and the reverse oligonucleotide sequence is synthesized; the synthesized forward oligonucleotide sequence is combined with The reverse oligonucleotide sequence is annealed and annealed to form a double-stranded oligonucleotide with sticky ends; (2) Ligate the double-stranded oligonucleotide into the expression vector lentiCRISPR v2 with the sequence shown in SEQ ID NO: 116 in the sequence listing, and digest the linearized vector with BsmB I restriction endonuclease to obtain a linearized vector carrying The recombinant expression vector lentiCRISPR v2-SLA-3 of sgRNA oligonucleotides is transformed into competent bacteria, the correct positive clones are screened and identified, and the positive clones are shaken to extract plasmids; (3) Use the expression vector lentiCRISPR v2-SLA-3, packaging plasmid and packaging cell line to package a pseudotyped lentivirus carrying both sgRNA and Cas9 targeting the SLA-3 gene; (4) Use the pseudotyped lentivirus to infect the target cells and further cultivate them; then collect the infected target cells, use their genomic DNA as a template to amplify the gene fragment containing the target sequence, and undergo denaturation, renaturation and enzymatic Cut to determine the knockout status of the SLA-3 gene. 7. the method for CRISPR-Cas9 specific knockout pig SLA-3 gene according to claim 6, is characterized in that, described packaging plasmid is plasmid pLP1, plasmid pLP2 and plasmid pLP/VSVG; Described packaging cell line is HEK293T cells. 8. the method for CRISPR-Cas9 specific knockout pig SLA-3 gene according to claim 6, is characterized in that, described target cell is pig PIEC cell; The gene fragment containing the target sequence is amplified using its genomic DNA as a template, and after denaturation, renaturation and enzyme digestion, the knockout situation of the SLA-3 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 SLA-3 gene to amplify the SLA-3 gene fragment containing the target sequence of the sgRNA, and simultaneously use the same primers to amplify the non-infected virus Genomic DNA from wild-type cells; (b) Purify the above-mentioned amplified SLA-3 gene fragment, and then mix the SLA-3 gene fragment from the target cell infected with the virus with the SLA-3 gene fragment from the wild-type cell in equal amounts, heat denature, and renature, Formation of hybrid DNA molecules; (c) cutting the annealed hybrid DNA molecule with Cruiser enzyme; (d) Electrophoresis to detect the digested products, and to detect the effect of target sequence-mediated SLA-3 gene knockout. 9. The recombinant expression vector lentiCRISPR v2-SLA-3 used in the method of CRISPR-Cas9 specific knockout pig SLA-3 gene, it is characterized in that, the sequence of the backbone vector of the recombinant expression vector is as SEQ in the sequence listing ID NO: 116; the target sequence carried is the target sequence of the sgRNA according to any one of claims 1-3, preferably the target sequence shown in SEQ ID NO: 4, 5 or 12 in the sequence listing. 10. Use of the sgRNA according to any one of claims 1-3 or the recombinant expression vector lentiCRISPR v2-SLA-3 according to claim 9 in the method for specifically knocking out the pig SLA-3 gene by CRISPR-Cas9.