WO2016197360A1 - Method for specific knockout of swine gfra1 gene using crispr-cas9 specificity, and sgrna used for specifically targeting gfra1 gene - Google Patents

Abstract

Provided is a method for using CRISPR-Cas9 specificity to knock out a swine GFRA1 gene, and an sgRNA used for specifically targeting the GFRA1 gene. The target sequence of the sgRNA for specifically targeting the GFRA1 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 GFRA1 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 GFRA1 gene.

Description

CRISPR-Cas9 specific knockdown of porcine GFRA1 gene and sgRNA for specific targeting of GFRA1 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 the porcine GFRA1 gene by CRISPR-Cas9 and an sgRNA for specifically targeting the GFRA1 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.

The traditional technical route achieves a strain of pigs that can be used for transplantation by reducing the difference in immunity between pigs and humans. In recent years, the use of organ-deficient pigs as a culture environment to produce organs composed of human cells has become a new idea. Through genetic engineering, it effectively interferes with the genes that control the development of pig's own organs, so that an organ is missing during development, which provides a key culture environment for the development of human cell organs.

The GFRA1 gene is currently known to be an essential gene in kidney development. GFRA1, the full name of the GDNF family receptor alpha 1, is highly conserved in evolution. GFRA1 acts as a receptor involved in the transmission of GDNF signaling, mediating signal feedback between the ureteric epithelium and the posterior renal interstitial, and is essential for ureteral buds entering the interstitial tissue. Deletion of the GFRA1 gene results in renal insufficiency or loss in neonatal rats. The use of GFRA1 gene knockout technology can prevent pigs from producing kidneys during development and provide a good development environment for human cell-derived kidneys. Accurate and efficient knockout of the GFRA1 gene in pigs is the first step.

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 GFRA1 gene by CRISPR technology can greatly improve the screening efficiency of genetically engineered pigs with GFRA1 deletion cells and renal developmental defects. 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 basis for knocking out the GFRA1 gene.

Summary of the invention

The object of the present invention is to provide a method for CRISPR-Cas9 specific knockdown of the porcine GFRA1 gene and an sgRNA for specifically targeting the GFRA1 gene.

According to a first aspect of the present invention, the present invention provides an sgRNA for specifically targeting a GFRA1 gene in a CRISPR-Cas9 specific knockout porcine GFRA1 gene, the sgRNA having the following characteristics:

(1) The target sequence of the sgRNA on the GFRA1 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, the target sequence conforming to the above rules is located in the sense strand or the antisense strand;

(2) The target sequence of the sgRNA on the GFRA1 gene is located in the 5 exon coding regions of the N-terminus of the GFRA1 gene, or a part of the target sequence is located at 5 N-terminal exons of the GFRA1 gene, and the rest Partially crossing the boundary with adjacent introns, located adjacent to the intron;

(3) The target sequence of the sgRNA on the GFRA1 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 50 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 or 4 in the Sequence Listing.

According to a second aspect of the present invention, the present invention provides a method of specifically knocking out a porcine GFRA1 gene using CRISPR-Cas9, 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) using the above expression vector carrying the sgRNA oligonucleotide and the Cas9 gene, a packaging plasmid, and a packaging cell line to package a pseudotype lentivirus carrying both sgRNA and Cas9 targeting the GFRA1 gene;

(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 GFRA1 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: 51 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 oligonucleotide having a cohesive terminus;

(2) A table in which the above double-stranded oligonucleotide is ligated into the sequence shown as SEQ ID NO: 51 in the Sequence Listing The linearized vector obtained by BsmB I restriction endonuclease was obtained from the vector lentiCRISPR v2, and the recombinant expression vector lentiCRISPR v2-GFRA1 carrying the sgRNA oligonucleotide was obtained, and the competent bacteria were transformed, and the correct positive clone was identified by screening. And positive clones were shaken and the plasmid was extracted;

(3) using the above expression vector lentiCRISPR v2-GFRA1, packaging plasmid and packaging cell line to package a pseudotype lentivirus carrying both sgRNA and Cas9 targeting the GFRA1 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 GFRA1 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 knockout of the GFRA1 gene is determined by denaturation, renaturation and enzymatic cleavage, specifically:

(a) using the genomic DNA of the target cell of the virus as a template, and using the upstream and downstream primers of the GFRA1 gene to amplify the GFRA1 gene fragment containing the target sequence of the above sgRNA, and simultaneously amplifying the genome of the wild-type cell not infected with the same primer. DNA

(b) purifying the amplified GFRA1 gene fragment, and then denaturation and renaturation of the GFRA1 gene fragment from the target cell infected with the virus and the GFRA1 gene fragment derived from the wild type cell 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 target sequence-mediated knockdown of GFRA1 gene.

According to a third aspect of the present invention, the present invention provides a recombinant expression vector lentiCRISPR v2-GFRA1 for use in a method for CRISPR-Cas9 specific knockout of a porcine GFRA1 gene, the sequence of the backbone vector of the recombinant expression vector being SEQ ID NO: ID NO: 51; 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 or 4 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-GFRA1 of the third aspect, in the method of CRISPR-Cas9 specific knockout of the porcine GFRA1 gene.

The invention specifically targets the CRISPR-Cas9 knock-out porcine GFRA1 gene, and successfully finds the sgRNA which specifically targets the GFRA1 gene, and uses the sgRNA of the present invention in the method of CRISPR-Cas9 specific knockout of the porcine GFRA1 gene, which can be rapidly , accurate, efficient and specific knockout of porcine GFRA1 The gene effectively solves the technical problem of constructing a GFRA1 gene knockout pig with a long 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 knockout effect of the target sequence of the enzyme digestion in the embodiment of the present invention, wherein M represents DNA Marker, and WT indicates that the PCR product of the wild type cell which has not been infected by virus infection and Cas9 is detected by the Cruiser enzyme digestion test. As a result, 1 and 4 respectively indicate the targeted cleavage effect of the target sequences No. 1 and No. 4 in Table 1 on the GFRA1 gene, and the arrow indicates a small fragment obtained by cleavage by the 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 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 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 sgRNA target sequence of Sus scrofa (pig) GFRA1 gene

1. GFRA1 gene sgRNA target sequence selection:

A suitable 20 bp oligonucleotide sequence was searched for as a target sequence in the exon region of the GFRA1 gene.

2. Design of sgRNA target sequence of GFRA1 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 CRISPR vector for constructing the GFRA1 gene

1. Synthesis of the above forward oligonucleotide sequence and reverse oligonucleotide sequence, renaturation forming a sticky end A double-stranded DNA fragment (ie, a double-stranded target sequence oligonucleotide, which may also be referred to as a double-stranded oligonucleotide).

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-GFRA1.

3. Obtaining pseudotyped lentivirus expressing GFRA1sgRNA

A CRISPR pseudotyped lentivirus expressing GFRA1sgRNA 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 GFRA1 gene

1. Lentivirus infection of the target cell:

A pseudotype lentivirus such as lentiCRISPR v2-GFRA1 is added to the cell culture medium of interest for infection and further culture.

2. Detection of GFRA1 gene knockout effect:

The target cell is collected, and the gene fragment containing the target sequence is amplified by using genomic DNA as a template, and the knockout of the GFRA1 gene is determined by denaturation, renaturation and restriction enzyme digestion.

5. Selection and identification of GFRA1 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 GFRA1 knockout.

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 sgRNA target sequence of Sus scrofa (porcine) GFRA1 gene

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 GFRA1 gene

For the GFRA1 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 GFRA1 gene, wherein N(20) represents 20 contiguous bases, wherein each N represents A or T Or C or G, the target sequence conforming to the above rules is located in the sense strand or the antisense strand;

(2) Selecting the coding region sequence of five exons near the N-terminus, the target sequence may be located in the coding region of the 5 exons of the N-terminus of the GFRA1 gene, or a part of the target sequence is located at the N-terminus of the GFRA1 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 results in the functional knockout of the GFRA1 gene, and the residual truncated sequence does not form. Functional protein;

(3) If there are multiple splicing bodies, the selection is performed in the shared exon coding region, and the sequence of 5 exon coding regions near the N-terminus is selected for the GFRA1 gene;

(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 GFRA1 gene.

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

Table 1 Target sequence collection

Numbering		sequence
			1	GTACTTCGCACTGCCGCTTC
2	GAAGCGGCAGTGCGAAGTAC
		3	TACTTCGCACTGCCGCTTCT
4	GTGCGAAGTACAGGGTCGCC
		5	AAGCGGCAGTGCGAAGTACA
6	GTACAGGGTCGCCAGGAACA
		7	GCACCAAGTACCGCACGCTG
8	CTCCTTTAGGCACTGATCGC
		9	CTGCCTCAGCGTGCGGTACT
10	CGAGTGCCGCAGCGCCATGG
		11	GGCGCTGCGGCACTCGTCCT
12	GGCCAGCGATCAGTGCCTAA
		13	GGACGAGTGCCGCAGCGCCA
14	CCGCACGCTGAGGCAGTGCG
		15	CTGTCGGCCGAGGTGAGCGG
16	CCACGCACTGCCTCAGCGTG
		17	CGCTGGCCTTCACACAGTCC

18	CACGCTGAGGCAGTGCGTGG
		19	CTTCAGCTTGACCTCGGGCC
20	GCCCGAGGTCAAGCTGAAGT
		twenty one	ACGCTGAGGCAGTGCGTGGC
twenty two	GACCAACTTCAGCTTGACCT
		twenty three	GGACCGCCTGGACTGTGTGA
twenty four	CGCCGCTCACCTCGGCCGAC
		25	TGTCGGCCGAGGTGAGCGGC
26	GCGGTCCCCGCCGCTCACCT
		27	ACCAACTTCAGCTTGACCTC
28	CTCCTGTCGGCCGAGGTGAG
		29	GGTGAGCGGCGGGGACCGCC
30	CAGCTTGACCTCGGGCCTGG
		31	GAGGCAGTGCGTGGCGGGCA
32	ACATGCTCCAGTAGATTCGC
		33	AGAACTGCCTGCGAATCTAC
34	TTACAACTGCCGCTGCAAGC
		35	TTTACAACTGCCGCTGCAAG
36	TGAGGGCCTCCATGGCGCTG
		37	GTAAAGCGACTTCTGTTTGA
38	GAGCATGTACCAGAGCCTGC
		39	CTGCAAGCGGGGTATGAAGA
40	TGTAAAGCGACTTCTGTTTG
		41	AGCATGTACCAGAGCCTGCA
42	CGGGTGGTCCCATTCATACC

43	ATTGTCTGATATTTTCCGGG
		44	GCTGTTAACTGGCTCATATG
45	CTGCTGTTAACTGGCTCATA
		46	TGCTGTTAACTGGCTCATAT
47	TCAGACAATCTGCTGTTAAC
		48	CAGATTGTCTGATATTTTCC
49	GCAGATTGTCTGATATTTTC
		50	CTGGCAGACGTTTTCCAGCA

2. Design of sgRNA target sequence of GFRA1 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 GFRA1 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 knockdown effect of the target sequence shown by the sequences No. 1 and No. 4 listed in Table 1 on the GFRA1 gene.

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

CACCGGTACTTCGCACTGCCGCTTC (SEQ ID NO: 52);

AAACGAAGCGGCAGTGCGAAGTACC (SEQ ID NO: 53).

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

CACCGGTGCGAAGTACAGGGTCGCC (SEQ ID NO: 54);

AAACGGCGACCCTGTACTTCGCACC (SEQ ID NO: 55).

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: 51 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 to the recovered vector fragment, and The next reaction system is prepared:

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-GFRA1 vector clone was seeded.

Example 3, obtaining a pseudotype lentivirus expressing GFRA1sgRNA

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-GFRA1; 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-GFRA1: 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

Cultured cells of the 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); lentiCRISPR v2-GFRA1 false for different target sequences (sequence 1 and sequence 4) Type lentivirus; 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-GFRA1 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 GFRA1 gene knockout effect

(1) Design upstream and downstream primers to amplify the GFRA1 gene fragment, wherein the upstream and downstream primer sequences are as follows:

CTCTCACTGGATGGAGCTGAACTTTG (SEQ ID NO: 56)

GCTCAGACCCTATAATTTCCTGCCAG (SEQ ID NO: 57).

The amplified fragment of interest consists of a sgRNA target sequence with a size of 465 bp. The position of the target sequence to both ends of the fragment is not less than 100 bp.

(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) Amplification of a GFRA1 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 target sequence-mediated knockdown of GFRA1 gene.

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. Since the mutant sample contains a part of the wild-type DNA component, it can form a hybrid molecule containing a local ring structure after being renatured.

As a result, as shown in Fig. 5, wild-type cells that had not been infected with the virus did not produce cleavage, and thus no small fragment was detected; and Sequence 1 and Sequence 4 were able to effectively target the GFRA1 gene to produce a cleavage, and thus the presence of a small fragment was detected, indicating Sequence 1 and Sequence 4 are capable of specifically knocking out the target sequence of the porcine GFRA1 gene by CRISPR-Cas9.

Example 5: Selection and identification of GFRA1 knockout monoclonal

1. Selection of monoclonals (based on sequence 1 and sequence 4 target sequences)

(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 GFRA1 knockout

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

(2) Amplification of the GFRA1 gene fragment of the monoclonal and wild-type cells according to the aforementioned method, and the amplified gene fragment contains the 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-GFRA1 pseudotype lentivirus infection target cell based on the target sequence shown in SEQ ID NO:1, and 6 monoclonal clones randomly selected from 100 single cells were detected by Cruiser enzyme electrophoresis, and 5 of them were single. Cloning can detect small fragments, indicating that gene knockout occurs, and the knockout efficiency can reach 83% or more, indicating that the target sequence shown in sequence 1 has a high target for knocking out the GFRA1 gene. The lentiCRISPR v2-GFRA1 pseudotype lentivirus infection target cell based on the target sequence shown in SEQ ID NO:4, four monoclonal cells randomly selected from 100 single cells were detected by Cruiser enzyme electrophoresis, and 4 of them were detected. By cutting small fragments, it is indicated that gene knockout occurs, and the knockout efficiency can reach 100%, indicating that the target sequence shown in sequence 4 has a high target for knocking out the GFRA1 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 GFRA1 gene in a CRISPR-Cas9-specific knockout porcine GFRA1 gene, characterized by:

   (1) The target sequence of the sgRNA on the GFRA1 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, the target sequence conforming to the rules is located in the sense strand or the antisense strand;

   (2) The target sequence of the sgRNA on the GFRA1 gene is located in the 5 exon coding regions of the N-terminus of the GFRA1 gene, or a part of the target sequence is located at 5 N-terminal exons of the GFRA1 gene, and the remaining portions span and adjacent The junction of introns, located in adjacent introns;

   (3) The target sequence of the sgRNA on the GFRA1 gene is unique.
2. The sgRNA for specifically targeting the GFRA1 gene according to claim 1, wherein the target sequence is the sequence shown by any one of SEQ ID NOS: 1 to 50 in the Sequence Listing.
3. The sgRNA for specifically targeting the GFRA1 gene according to claim 1, wherein the target sequence is the sequence shown by SEQ ID NO: 1 or 4 in the Sequence Listing.
4. A method for specifically knocking out a porcine GFRA1 gene using 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 GFRA1 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 GFRA1 gene.
5. The CRISPR-Cas9-specific knock-out porcine GFRA1 gene according to claim 4, wherein the expression vector is a vector of the sequence of SEQ ID NO: 51 in the Sequence Listing.
6. A method of specifically knocking out a porcine GFRA1 gene by a CRISPR-Cas9 according to claim 4 or 5, wherein the method comprises the steps of:

   (1) A forward oligonucleotide sequence is synthesized by adding a CACCG sequence to the 5'-end of the target sequence of the sgRNA according to any one of claims 1 to 3; The 5'-end of the complementary sequence corresponding to the target sequence of the sgRNA is added to the AAAC sequence, and the 3'-end plus C is synthesized to obtain a reverse oligonucleotide sequence; the synthesized forward oligonucleotide sequence is The reverse oligonucleotide sequence is annealed and renatured 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: 51 in the sequence listing by BsmB I restriction endonuclease to obtain a vector. The recombinant expression vector lentiCRISPR v2-GFRA1 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-GFRA1, a packaging plasmid, and a packaging cell line to package a pseudotype lentivirus carrying both sgRNA and Cas9 targeting the GFRA1 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 GFRA1 gene.
7. The CRISPR-Cas9-specific knockout porcine GFRA1 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 knockout of the GFRA1 gene is determined by denaturation, renaturation and restriction enzyme digestion, specifically:

   (a) amplifying the GFRA1 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 GFRA1 gene. Genomic DNA;

   (b) purifying the amplified GFRA1 gene fragment, and then denaturation and renaturation of the GFRA1 gene fragment from the target cell infected with the virus and the GFRA1 gene fragment derived from the wild type cell 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 target sequence-mediated knockdown of GFRA1 gene.
9. The recombinant expression vector lentiCRISPR v2-GFRA1 used in the method of CRISPR-Cas9 specific knockout of the porcine GFRA1 gene, characterized in that the sequence of the backbone vector of the recombinant expression vector is as shown in SEQ ID NO: 51 in the sequence listing. The target sequence carried by the sgRNA of any one of claims 1 to 3, preferably the target sequence of SEQ ID NO: 1 or 4 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-GFRA1 according to claim 9 in a method of CRISPR-Cas9 specific knockout of the porcine GFRA1 gene.

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