CN106566838A - MiR-126 full-length gene knockout kit based on CRISPR-Cas9 technology and application thereof - Google Patents

Abstract

The invention discloses a miR-126 full-length gene knockout kit based on CRISPR-Cas9 technology and its application. In the present invention, the upstream and downstream CRISPR-Cas9 target sequence of the miR-126 gene is preferred, and a sgRNA single strand targeting the target sequence is designed and synthesized, and constructed into a vector. By transfecting the 293T cell line, the sgRNA and trRNA form a specific recognition structure, thereby guiding Cas9 The enzyme specifically cuts the corresponding sequences at both ends of the miR-126 gene, and continues drug screening to obtain a full-length miR-126 knockout cell line; after sequencing and verification of the drug-screening cell line, a preferred combination of upstream and downstream sgRNAs is obtained, and the knockout of this combination The efficiency is as high as more than 90%. This kit can be used to knock out the full-length gene of miR-126 specifically for various types of cell lines such as 293T, lung cancer cell line A549, and vascular endothelial cell line HUVEC.

Description

A miR-126 full-length gene knockout kit based on CRISPR-Cas9 technology and its application

technical field

The invention belongs to the field of genetic engineering, and relates to a miR-126 gene knockout kit, in particular to a kit capable of specifically knocking out the full-length gene sequence of miR-126 based on CRISPR-Cas9 technology. In addition, the invention also discloses the application of the kit.

Background technique

microRNA (microRNA) is a kind of non-coding single-stranded RNA molecule encoded by endogenous genes with a length of about 22 nucleotides, which is widely distributed in viruses, plants and higher mammals, and has many important functions in cells. the regulating effect. Each microRNA can have multiple target genes, and several microRNAs can also regulate the same gene. This complex regulatory network can not only regulate the expression of multiple genes through a microRNA, but also finely regulate the expression of a gene through the combination of several microRNAs. Therefore, the gene function exploration and application development of microRNAs have always been a research hotspot.

miR-126 (microRNA126, microRNA126) is located in the intron of the epidermal growth factor like 7 (epidermal growth factor like 7, EFGL7) gene on human chromosome 9q34.3. miR-126 is a microRNA specifically expressed in vascular endothelial cells, and is highly expressed in tissues rich in blood vessel formation, such as heart, lung, and kidney. It can regulate Spred-1 (Sprouty-related, EVH1 domain-containing protein 1, Sprouty-related EVH1 domain-containing protein 1), VCAM-1 (Vascular cell adhesion protein 1, vascular cell adhesion molecule-1), HoxA9 (homeobox A9, homeobox gene A9), v-Crk (Sarcoma Virus CT10 regulator of kinase), EGFL-7 and VEGF (vascular endothelial growth factor, vascular endothelial growth factor) and other genes are involved in the regulation of vascular development Physiological and pathophysiological processes of blood vessels, such as angiogenesis and vascular inflammation. Other studies have found that miR-126 expression is suppressed in gastrointestinal, reproductive tract cancers, breast, thyroid, lung, and other cancers. Down-regulation of miR-126 can induce cancer cell proliferation, migration, invasion and affect cell survival. There is a correlation between down-regulation of miR-126 and poor survival of various cancers. These research findings provide potential targets for the judgment of metastasis prevention of cancers of the gastrointestinal tract, reproductive tract, breast, thyroid, lung, etc. direction, suggesting the importance and necessity of miR-126 research.

Because the microRNA sequence is short and its precursor has a stem-loop structure, the classic RNA interference (RNAi) method is not suitable for the functional study of microRNA down-regulated expression. At present, the commonly used method of microRNA research is to apply biological technology in in vivo cell models or in vitro animal/plant models to block the sequence of microRNA or cause gene deletion mutations to simulate the weakening or removal of the negative regulatory effect caused by the loss of microRNA function. There are two main types of technical methods to study its functional mechanism:

The first type of method is to design and synthesize the complementary closed sequence of microRNA. The complementary sequence competes with the original specific binding mRNA of microRNA, weakening the binding ability and regulatory effect of microRNA to specific mRNA. Since the complementary sequence blocks the microRNA in the form of base complementarity, it does not directly lead to the degradation or downregulation of the expression of the microRNA, so the expression level of the microRNA does not change, and there is no direct technical method to quantify the blocking efficiency of the microRNA function Detection cannot directly prove that the function of microRNA has been shielded, but can only indirectly indicate that the function of microRNA is inhibited by detecting the possible regulation of downstream gene changes of microRNA. Therefore, this method has a great impact on the authenticity and effectiveness of microRNA function research. .

The second type of method is to use transcription activator-like effector nuclease (transcription activator-like effector nuclease, TALEN) TALEN technology, through the DNA recognition module to target the TALEN element to a specific DNA site and bind, and then in the action of FokI nuclease By completing the cleavage at a specific site, the microRNA can be cleaved and modified at the DNA level, simulating the loss of microRNA function. However, the knockout of the target gene by TALEN technology relies on the amino acid polymer module sequence combined with DNA. The assembly of a single TALEN module requires a large number of molecular cloning and sequencing operations, which is very cumbersome. It takes more than 2 months, and it takes more than 4 months to screen a cell line with stable gene knockout. The whole process is time-consuming and costly.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats/cas, clustered, regularly spaced short palindromic repeats) technology, also known as CRISPR/Cas9 (CRISPR associated protein 9, CRISPR-associated protein 9) nuclease technology is the latest emerging A genome editing tool that enables RNA-guided DNA recognition and editing. Compared with the gene technology tools introduced above, CRISPR technology is easier to operate and has stronger scalability.

CRISPR/Cas9 was first discovered in the natural immune system of bacteria. Its main function is to fight against invading viruses and foreign DNA, and it is the acquired immune defense mechanism of bacteria and archaea. In this system, crRNA (CRISPR-derived RNA, CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA, trans-activating RNA) through base pairing to form a double-stranded RNA, and this tracrRNA/crRNA binary complex guides Cas9 The nuclease protein cuts the double-stranded DNA at the target site of the crRNA guide sequence, thereby achieving the purpose of cutting or modifying the genomic DNA.

In order to realize the application of the CRISPR/Cas9 system in mammals, scientists have modified CRISPR/Cas9 to fuse and express the tracrRNA/crRNA binary complex to form a single-stranded guide RNA. In the actual design, only a 20nt (base) RNA sequence that recognizes the target sequence is designed and synthesized and called sgRNA (small guide RNA, small guide RNA), and other parts of the guide RNA are used as sgRNA expression vectors because the sequence remains unchanged. The vector sequence above exists. Therefore, the widely used CRISPR/Cas9 system has become a technology for targeted gene editing by Cas9 nuclease under the guidance of guide RNA containing sgRNA.

The most common site-directed shear editing technology of CRISPR/Cas9 technology is to design sgRNA for a single site to guide Cas9 to perform directional shearing on the target sequence (for example, "a gene knockout based on CRISPR-Cas9" disclosed in Chinese invention patent application CN105112445A technology miR-205 gene knockout kit"), the inherent non-homologous end-joining pathway (NHEJ) repair process in cells after cleavage will introduce random insertion and deletion gene mutations, this repair process is not under human control, resulting in Various unnecessary and inaccurate sequence insertions and deletions often produce ambiguous or invalid cell phenotypes, adversely affecting research.

At present, there is no report about using the CRISPR/Cas9 system to specifically knock out the full-length gene sequence of miR-126.

Contents of the invention

One of the technical problems to be solved by the present invention is to provide a kit for knocking out the miR-126 gene based on CRISPR/Cas9 gene editing technology, which enables CRISPR/Cas9 to simultaneously target both ends of the miR-126 gene coding sequence. Site-specific cutting can greatly avoid the possibility of random mutations introduced by the non-homologous end-joining pathway NHEJ caused by the traditional design of sgRNA for a single site to guide Cas9 to perform directional cutting on the target sequence, which is very beneficial To produce a single and obvious cell phenotype for the complete deletion of the gene, the kit can efficiently, quickly, accurately and stably realize the complete knockout of the full-length coding sequence of miR-126 at the genome level.

The second technical problem to be solved by the present invention is to provide the application of the kit, which can be applied to miR- 126 gene knockout, these cell lines can be further used to construct animal models by transplanting tumors in nude mice, which provide a good tool for the research of miR-126-related pathways and drug development in vivo and in vitro experimental models.

The present invention optimizes a certain number of upstream and downstream CRISPR-Cas9 target sequences of the miR-126 gene by designing target software, designs and synthesizes sgRNA single strands for the upstream and downstream target sequences respectively, constructs them into plasmid vectors, and transfects 293T cells sgRNA and trRNA (trans-activating RNA) form a specific recognition structure, thereby guiding the Cas9 enzyme to specifically cut the corresponding sequences at both ends of the miR-126 gene, and continue drug screening to obtain a full-length miR-126 gene knockout cell line . Cellular DNA was extracted, amplified by PCR, and confirmed and verified using the gold standard technology for gene knockout detection at the DNA level—generation sequencing technology, and two effective upstream and downstream sgRNA combinations for miR-126 gene knockout CRISPR-Cas9 were obtained , Fluorescent quantitative PCR technology showed that the knockout efficiency of miR-126 molecules was as high as 90%. The application kit was constructed to detect 293T, lung cancer cell line A549, breast cancer cell line T47D, and vascular endothelial cell line HUVEC, etc. Stable, accurate and specific knockout of the full-length miR-126 gene sequence was carried out in several types of cell lines.

In order to solve the problems of the technologies described above, the present invention adopts the following technical solutions:

In one aspect of the present invention, a miR-126 full-length gene knockout kit based on CRISPR-Cas9 technology is provided,

The kit includes a combination of sgRNA plasmids consisting of plasmid pSpCas9(BB)-2A-Puro-sgRNA1 and plasmid pSpCas9(BB)-2A-Puro-sgRNA3; the plasmid pSpCas9(BB)-2A-Puro-sgRNA1 contains a Complementary DNA sequence, which can be transcribed into sgRNA with a specific recognition sequence such as the target sequence of the miR-126 full-length gene shown in SEQ ID NO.2. The sgRNA can form a specific recognition structure with trRNA, so as to guide Cas9 The enzyme specifically cuts the corresponding sequence of the miR-126 gene; the plasmid pSpCas9(BB)-2A-Puro-sgRNA3 contains a complementary DNA sequence that can be transcribed into a specific recognition sequence as shown in SEQ ID NO.4 The sgRNA of the target sequence of the full-length miR-126 gene shown, the sgRNA can form a specific recognition structure with trRNA, so as to guide the Cas9 enzyme to specifically cut the corresponding sequence of the miR-126 gene;

Alternatively, the kit includes a combination of sgRNA plasmids consisting of plasmid pSpCas9(BB)-2A-Puro-sgRNA2 and plasmid pSpCas9(BB)-2A-Puro-sgRNA5; said plasmid pSpCas9(BB)-2A-Puro-sgRNA2, Containing a complementary DNA sequence, the DNA sequence can be transcribed into a sgRNA with a specific recognition sequence such as the target sequence of the miR-126 full-length gene shown in SEQ ID NO.3, and the sgRNA can form a specific recognition structure with trRNA, so that Guide the Cas9 enzyme to specifically cut the corresponding sequence of the miR-126 gene; the plasmid pSpCas9(BB)-2A-Puro-sgRNA5 contains a complementary DNA sequence that can be transcribed into a specific recognition sequence such as SEQ ID NO. The sgRNA of the target sequence of the full-length miR-126 gene shown in 6, the sgRNA can form a specific recognition structure with the trRNA, so as to guide the Cas9 enzyme to specifically cut the corresponding sequence of the miR-126 gene.

As a preferred technical solution of the present invention, the complementary DNA sequences contained in the plasmid pSpCas9(BB)-2A-Puro-sgRNA1 are SEQ ID NO.7 and SEQ ID NO.8.

As a preferred technical solution of the present invention, the complementary DNA sequences contained in the plasmid pSpCas9(BB)-2A-Puro-sgRNA3 are SEQ ID NO.11 and SEQ ID NO.12.

As a preferred technical solution of the present invention, the complementary DNA sequences contained in the plasmid pSpCas9(BB)-2A-Puro-sgRNA2 are SEQ ID NO.9 and SEQ ID NO.10.

As a preferred technical solution of the present invention, the complementary DNA sequences contained in the plasmid pSpCas9(BB)-2A-Puro-sgRNA5 are SEQ ID NO.15 and SEQ ID NO.16.

As a preferred technical scheme of the present invention, the sgRNA plasmid combination composed of the plasmid pSpCas9(BB)-2A-Puro-sgRNA1 and the plasmid pSpCas9(BB)-2A-Puro-sgRNA3, the plasmid pSpCas9(BB)-2A-Puro - The sgRNA plasmid combination consisting of sgRNA2 and plasmid pSpCas9(BB)-2A-Puro-sgRNA5 is constructed by a method comprising the following steps:

Step 1: Design and optimize a certain number of miR-126 gene upstream and downstream CRISPR-Cas9 target sequences by designing the target software, as follows:

upstream:

sgRNA-1: TAATGTCCCGTCGCCAGCGG, as shown in SEQ ID NO.2;

sgRNA-2: GCCACGCCTCCGCTGGCGAC, as shown in SEQ ID NO.3;

Downstream:

sgRNA-3: TTCTCAGCGGCGTTTTCGATG, as shown in SEQ ID NO.4;

sgRNA-4: GAGTAATAATGCGCCGTCCA, as shown in SEQ ID NO.5;

sgRNA-5: TTTCGATGCGGTGCCGTGGA, as shown in SEQ ID NO.6;

Step 2, respectively designing and synthesizing sgRNA fragments targeting upstream and downstream target sequences, and constructing them into plasmid vectors; the sequences of the sgRNA fragments are as follows:

sgRNA-1F: ccacgTAATGTCCCGTCGCCAGCGG, as shown in SEQ ID NO.7;

sgRNA-1R: aaacCCGCTGGCGACGGGACATTAc, as shown in SEQ ID NO.8;

sgRNA-2F: ccacGCCACGCCTCCGCTGGCGAC, as shown in SEQ ID NO.9;

sgRNA-2R: aaac GTCGCCAGCGGAGGCGTGGC, as shown in SEQ ID NO.10;

sgRNA-3F ccacgTCTCAGCGGCGTTTTCGATG, as shown in SEQ ID NO.11;

sgRNA-3R aaacCATCGAAAACGCCGCTGAGAC, as shown in SEQ ID NO.12;

sgRNA-4F ccacGAGTAATAATGCGCCGTCCA, as shown in SEQ ID NO.13;

sgRNA-4R aaacTGGACGGCGCATTATTACTC, as shown in SEQ ID NO.14;

sgRNA-5F ccacgTTTCGATGCGGTGCCGTGGA, as shown in SEQ ID NO.15;

sgRNA-5R aaacTCCACGGCACCGCATCGAAAc, as shown in SEQ ID NO.16;

Step 3: By transfecting the 293T cell line, sgRNA and trRNA form a specific recognition structure, thereby guiding the Cas9 enzyme to specifically cut the corresponding sequences at both ends of the miR-126 gene, and continue drug screening to obtain the miR-126 full-length gene knockout cell line;

Step 4, determine and verify by extracting cell DNA, PCR amplification, and sequencing to obtain two sets of optimized miR-126 gene knockout CRISPR-Cas9 sgRNA plasmid combinations, namely the plasmid pSpCas9(BB)-2A-Puro- The sgRNA plasmid combination composed of sgRNA1 and plasmid pSpCas9(BB)-2A-Puro-sgRNA3, and the sgRNA plasmid combination composed of the plasmid pSpCas9(BB)-2A-Puro-sgRNA2 and plasmid pSpCas9(BB)-2A-Puro-sgRNA5 .

As a preferred technical solution of the present invention, in step 2, the construction into a plasmid vector specifically includes the following steps:

(1) Using the pSpCas9(BB)-2A-Puro plasmid as the starting vector, perform single enzyme digestion with Bbs I, and purify the linearized vector;

(2) Dilute and anneal the synthesized sgRNA fragments;

(3) Using T4DNA ligase to connect the linearized pSpCas9(BB)-2A-Puro to the annealed sgRNA fragment, and add all the ligated products to E. coli DH5α competent cells for transformation;

(4) Using the colony PCR method to identify the single colony grown on the plate coated with the DH5α competent cell bacterial solution after transformation, and obtain the expression plasmid.

In another aspect of the present invention, an application of the above-mentioned kit in preparing a cell line miR-126 gene knockout product is provided. The cell lines include tool cell line 293T, tumor cell lines (eg, lung cancer cell line A549, breast cancer cell line T47D) and endothelial cell HUVEC lines.

Compared with the prior art, the present invention has the following beneficial effects: the miR-126 full-length gene knockout kit based on the CRISPR-Cas9 technology of the present invention mainly uses the CRISPR/Cas9 system to knock out the miR-126 target gene. This is the first time that the full-length gene sequence of miR-126 has been specifically knocked out using the CRISPR/Cas9 system. The present invention has the following advantages:

1. To overcome the drawbacks of the traditional closed-sequence method for studying microRNA functions, the results of miR-126 knockout can be monitored quantitatively, and the knockout efficiency of miR-126 molecules is as high as 90% or more;

2. It overcomes the time-consuming and cumbersome defects of TALEN technology, accelerates the cell screening test and shortens it to 1 week, and stably constructs cell line experiments to 2 months; the construction steps and experimental operations are simple, and the cost of reagents is reduced;

3. On the basis of CRISPR-Cas9 technology, a technical extension design was made, and sgRNA was designed at both upstream and downstream ends of the miR-126 gene sequence, and simultaneous fixed-point cutting of two sites was performed, realizing the detection of the entire miR-126 at the DNA level. 126 gene sequences were completely knocked out (see Figure 15), and the first-generation sequencing results showed that the experimental results can be repeated in a variety of cells, and the stability is good. The present invention greatly avoids the possibility of random mutations introduced by the non-homologous end joining pathway NHEJ caused by the traditional method of designing sgRNA for a single site to guide the Cas9 target sequence for directional cutting, and can accurately realize the full-length coding sequence of microRNA Complete knockout at the genome level.

Description of drawings

1 is a schematic diagram of the construction of the pSpCas9(BB)-2A-Puro-sgRNA carrier verified by PCR method in Example 1 of the present invention; wherein, passages 1-5 are the positive clone PCR results of the construction of the pSpCas9(BB)-2A-Puro-sgRNA carrier; Lane M is the DNA molecular weight standard.

Figure 2 is a schematic diagram of the knockout results of pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A-Puro-sgRNA3 paired sgRNA combinations in 293T in Example 2 of the present invention; wherein, channels 3, 4, and 5 are pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A-Puro-sgRNA3 combined knockout PCR positive results; Lanes 1 and 2 are pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A -Puro-sgRNA5 combination knockout PCR negative result, channel 6 is the PCR result of control 293T cells; channel M is the DNA molecular weight standard.

Fig. 3 is a schematic diagram of the knockout verification sequencing results of pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A-Puro-sgRNA3 paired sgRNA combination in 293T in Example 2 of the present invention.

Figure 4 is a schematic diagram of the knockout results of pSpCas9(BB)-2A-Puro-sgRNA2 and pSpCas9(BB)-2A-Puro-sgRNA5 paired sgRNA combination in 293T in Example 2 of the present invention; wherein, channel 1 is pSpCas9(BB) -Positive result of 2A-Puro-sgRNA2 and pSpCas9(BB)-2A-Puro-sgRNA5 combined knockout PCR, lanes 2, 3, 4, 5, 6, 7, 8 are pSpCas9(BB)-2A-Puro-sgRNA2 Combined with pSpCas9(BB)-2A-Puro-sgRNA4 knockout PCR negative results, channel 9 is the PCR result of the control 293T cells; channel M is the DNA molecular weight standard.

Fig. 5 is a schematic diagram of the knockout verification sequencing results of pSpCas9(BB)-2A-Puro-sgRNA2 and pSpCas9(BB)-2A-Puro-sgRNA5 paired sgRNA combinations in 293T in Example 2 of the present invention.

Figure 6 is a schematic diagram of the PCR verification knockout results of monoclonal cells isolated from 293T by drug screening after transfection in Example 2 of the present invention; wherein, channel 1 is the positive PCR result of 293T knockout miR-126 monoclonal cells, and channel 2 It is the PCR result of the control 293T cells without knockout; channel M is the DNA molecular weight standard.

Fig. 7 is a schematic diagram of PCR fragment sequencing results of knockout-positive monoclonal cells isolated from 293T after transfection by drug screening in Example 2 of the present invention.

Figure 8 is a schematic diagram of the fluorescence quantitative qPCR detection results of miR-126 knockout 293T stable strain in Example 2 of the present invention; wherein, 293Tblank is the fluorescence quantitative qPCR result of 293T control cells without miR-126 knockout, and 293T cloneD2 is 293T knockout miR Fluorescent quantitative qPCR results of -126 positive clone D2 cells.

Figure 9 is a schematic diagram of the knockout results of A549 monoclonal cells isolated by drug screening after transfection in Example 3 of the present invention; wherein, lanes 1, 2, 3, 4, 5, and 6 are A549 knockout miR- After 126, the PCR positive result, channel 7 is the PCR result of the control A549 cells without miR-126 knockout; channel M is the DNA molecular weight standard.

Fig. 10 is a schematic diagram of the PCR fragment sequencing results of the knockout-positive monoclonal cells isolated by drug screening after transfection in Example 3 of the present invention.

Figure 11 is a schematic diagram of the knockout results of monoclonal cells obtained by drug screening of HUVEC after transfection in Example 4 of the present invention; wherein, channel 2 is the positive PCR result of HUVEC knockout of miR-126, channels 1, 3, 4 is the PCR result of miR-126 not knocked out in control HUVEC cells; channel M is the DNA molecular weight standard.

Fig. 12 is a schematic diagram of PCR fragment sequencing results of the knockout miR-126-positive monoclonal cells isolated by drug screening after transfection in Example 4 of the present invention.

Figure 13 is a schematic diagram of the knockout results of T47D cells after drug screening after transfection in Example 5 of the present invention; wherein, channels 1 and 2 are the positive PCR results of miR-126 knockout in T47D cell samples, and channels 3 and 4 are The PCR results of control T47D cells without miR-126 knockout; channel M is the DNA molecular weight standard.

Fig. 14 is a schematic diagram of PCR fragment sequencing results of cells positive for miR-126 knockout after transfection and T47D drug screening in Example 5 of the present invention.

Figure 15 is a schematic diagram of complete knockout of miR-126 gene sequence.

detailed description

Below in conjunction with specific embodiment further elaborates this invention. It should be understood that the specific embodiments described herein are presented by way of example and not as limitations of the invention. The principal characteristics of this invention can be employed in various embodiments without departing from the scope of the invention.

Example 1. Vector construction

(1) miR-126 target design

For the miR-126 gene (gene name miR-126, gene ID number: 406913, see http://www.ncbi.nlm.nih.gov/gene/406913 for gene details), download miR-126 from this website Genome sequence:

5'-CGCTGGCGACGGGACATTATTACTTTTGGTACGCGCTGTGACACTTCAAACTCGTACCGTGAGTAATAATGCGCCGTCCACGGCA-3', as shown in SEQ ID NO.1.

Use the online software Feng Zhang lab's Target Finder (http://crispr.mit.edu/) to design sgRNA, input the miR-126 genome sequence and its upstream and downstream 50bp sequences, set and retrieve several sgRNA sequences, and analyze the sgRNA in The location on the gene sequence and the off-target (off-target) information of the sgRNA, from which the optimal 2 upstream target sequences and 3 downstream target sequences are respectively selected, as follows:

upstream:

sgRNA-1: TAATGTCCCGTCGCCAGCGG, as shown in SEQ ID NO.2;

sgRNA-2: GCCACGCCTCCGCTGGCGAC, as shown in SEQ ID NO.3;

Downstream:

sgRNA-3: TTCTCAGCGGCGTTTTCGATG, as shown in SEQ ID NO.4;

sgRNA-4: GAGTAATAATGCGCCGTCCA, as shown in SEQ ID NO.5;

sgRNA-5: TTTCGATGCGGTGCCGTGGA, as shown in SEQ ID NO.6;

(2) Synthesis of sgRNA fragments

According to the information of the enzyme cutting site Bbs I of the vector pSpCas9(BB)-2A-Puro (purchased from Addgene) into which the sgRNA is to be cloned, after obtaining the complementary sequence of the above sgRNA sequence, add the Bbs I restriction endonuclease Corresponding cohesive ends. The following sequence is obtained:

sgRNA-1F: ccacgTAATGTCCCGTCGCCAGCGG, as shown in SEQ ID NO.7;

sgRNA-1R: aaacCCGCTGGCGACGGGACATTAc, as shown in SEQ ID NO.8;

sgRNA-2F: ccacGCCACGCCTCCGCTGGCGAC, as shown in SEQ ID NO.9;

sgRNA-2R: aaac GTCGCCAGCGGAGGCGTGGC, as shown in SEQ ID NO.10;

sgRNA-3F ccacgTCTCAGCGGCGTTTTCGATG, as shown in SEQ ID NO.11;

sgRNA-3R aaacCATCGAAAACGCCGCTGAGAC, as shown in SEQ ID NO.12;

sgRNA-4F ccacGAGTAATAATGCGCCGTCCA, as shown in SEQ ID NO.13;

sgRNA-4R aaacTGGACGGCGCATTATTACTC, as shown in SEQ ID NO.14;

sgRNA-5F ccacgTTTCGATGCGGTGCCGTGGA, as shown in SEQ ID NO.15;

sgRNA-5R aaacTCCACGGCACCGCATCGAAAc, as shown in SEQ ID NO.16.

The above sequences were sent to Thermofisher to synthesize single-stranded nucleotide chains.

(3) The sgRNA fragment is connected with the expression vector and transformed

1 μg of pSpCas9(BB)-2A-Puro was digested with Bbs I (purchased from NEB Company, Cat. DP204) to purify the linearized vector.

Dilute the dry powder of the F and R chains of each group of sgRNA synthesized to 100 μM, take 1 μL of each solution and mix it in a PCR tube, and gradually drop from 95 °C to 22 °C at 1.5 °C per minute in the PCR instrument.

Use T4 DNA ligase (purchased from Takara, Cat. No. 6022Q) to connect 1 μL of linearized pSpCas9(BB)-2A-Puro to 0.5 μL of the annealed sgRNA fragment. The connection system is shown in Table 1:

Table 1

sgRNA 0.5μL Linearized pSpCas9(BB)-2A-Puro 1μL DNAsolution I 5μL ddH ₂ O up to 10 μL

After ligation at 16°C for 15 minutes, all the ligation products were added to 100 μL E. coli DH5α competent cells (pfu≥10 ⁸ ), placed on ice for 30 minutes, then heat-shocked in a water bath at 42°C for 90 seconds, and placed on ice for 5 minutes. Add 1mL LB liquid medium (Luria-Bertani medium) to the ultra-clean bench, culture with shaking at 37°C for 1 hour, take 500μL and spread it on the LB solid medium containing Amp (Ampicillin, ampicillin) and wait for the liquid to absorb After completion, incubate overnight at 37°C.

(4) Identification after transformation

The colony PCR method was used to identify the single colony grown on the plate coated with DH5α competent cell bacterial liquid after transformation. The primers for colony PCR identification are as follows:

Primer F: 5'GACTATCATATGCTTACCG 3', as shown in SEQ ID NO.17;

Primer R: 5'CCAAGTGGGCAGTTTACC 3', as shown in SEQ ID NO.18;

The bacteria solution PCR system is shown in Table 2:

Table 2

Element volume 10×buffer 2.5μL primer F (10μM) 0.2 μL primer R (10μM) 0.2 μL dNTPs (2.5mM) 2μL rTaq enzyme (50U/μL) 0.5μL colony dip a little ddH ₂ O up to 25μL

The PCR program was 95°C for 10 min; 95°C for 20 sec, 55°C for 30 sec, and 72°C for 30 sec, a total of 35 cycles; 72°C for 10 min. The obtained PCR product is detected by 1% agarose gel electrophoresis, and the single colony with about 100 bp amplified fragments is a single colony containing positive clones (as shown in Figure 1, the PCR product fragment size is the same as the 100 bp fragment of the DNA molecular weight standard similar in size, and the 1000 bp fragment is the brightest fragment of this molecular weight standard). Inoculate a single colony of positive clones into 2 mL of LB liquid medium, shake culture overnight at 37°C, and take 1 mL of bacterial liquid for seed preservation, and send the remaining 1 mL of bacterial liquid to a sequencing company (Shanghai Meiji Biomedical Technology Co., Ltd.) for sequencing verification. Blast analysis of the obtained sequencing results showed that the sgRNA fragment had been cloned into the pSpCas9(BB)-2A-Puro plasmid with the same sequence and could be used for subsequent experiments. The vectors with sgRNA were named pSpCas9(BB)-2A-Puro-sgRNA1, pSpCas9(BB)-2A-Puro-sgRNA2, pSpCas9(BB)-2A-Puro-sgRNA3, pSpCas9(BB)-2A-Puro-sgRNA3, pSpCas9(BB)-2A-Puro- sgRNA4 and pSpCas9(BB)-2A-Puro-sgRNA5.

Example 2. Plasmid transfection and identification of miR-126 knockout activity in 293T cells and construction of 293T knockout miR-126 cell model

(1) Plasmid extraction

The Escherichia coli DH5α of the pSpCas9(BB)-2A-Puro plasmid respectively comprising sgRNA-1, sgRNA-2, sgRNA-3, sgRNA-4, and sgRNA-5 fragments verified by sequencing in Example 1 were each inoculated in 5 mL In LB liquid medium, culture overnight at 37°C with shaking. Plasmid extraction was performed on these bacterial liquids using an endotoxin-free mini-plasmid extraction kit (the kit was produced by Omega Company, the article number is D6948-02). The extracted plasmid is then precipitated with absolute ethanol in an ultra-clean bench, and then dissolved in sterile water for aseptic treatment of the plasmid.

(2) 293T cells were transfected with a pair of pSpCas9(BB)-2A-Puro-sgRNA plasmids containing sgRNA

1) 293T cell culture

Spread 293T cells in a 24-well plate at a density of 3×10 ⁵ cells/well to ensure that the cells in each well are in a good growth state and have a similar density. After the cells are monolayer and in mid-logarithmic phase, the cell confluence reaches about 80%. transfection.

2) Prepare the transfection complex:

According to the pairing in Table 3, a pair of pSpCas9(BB)-2A-Puro-sgRNA plasmids containing sgRNA were transfected into 293T cells by Lipofectamine 2000 liposome reagent.

Table 3 Pairing table of upstream and downstream plasmids containing sgRNA

serial number Upstream sgRNA plasmid Downstream sgRNA plasmid 1 pSpCas9(BB)-2A-Puro-sgRNA1 pSpCas9(BB)-2A-Puro-sgRNA3 2 pSpCas9(BB)-2A-Puro-sgRNA1 pSpCas9(BB)-2A-Puro-sgRNA5 3 pSpCas9(BB)-2A-Puro-sgRNA2 pSpCas9(BB)-2A-Puro-sgRNA4 4 pSpCas9(BB)-2A-Puro-sgRNA2 pSpCas9(BB)-2A-Puro-sgRNA5

Take 2 sterile EP tubes, dilute 0.8 μg of plasmid and 2 μL of Lipofectamine 2000 in 50 μL of serum-free and antibiotic-free Opti-MEM medium respectively, let stand at room temperature for 5 minutes, mix the diluted plasmid and Lipofectamine 2000, and obtain a total of 100 μL of transfection complex. After mixing gently, incubate at room temperature for 20 minutes.

3) Cell transfection:

Add the above transfection complex to the corresponding 24-well plate, gently shake the cell culture plate back and forth, put the cells back into the 37°C 5% CO ₂ incubator and continue to culture for 6 hours, then replace the medium with complete medium, continue Cells were cultured in a 37°C 5% _CO2 incubator.

4) Drug sieve

24 hours after transfection, the medium was replaced with complete medium containing 2 μg/mL puromycin. The drug-containing complete medium was replaced every 24 hours for three consecutive days. Observe the green fluorescent signal under the microscope every day and record the cell state.

(3) CRISPR/Cas9 knockout efficiency verification

1) PCR sequencing method to verify the knockout efficiency at the genome level

The 293T cells after drug screening and the 293T cells without transfection were collected, and the ultra-micro sample genotype identification kit (Nanjing Yaoshunyu Biotechnology Co., Ltd., KC-101) was used for sample processing, and as shown in Table 4 PCR primers for PCR amplification identification.

Table 4

*Primer sequence (5'--3') *Primer name GAGGGAGGATAGGTGGGTTC, as shown in SEQ ID NO.19 mir126-test-Fw AGGCAGAGCCAGAAGACTCA, as shown in SEQ ID NO.20 mir126-test-Rv GCACTGGAATCTGGGCGGAAGG, as shown in SEQ ID NO.21 mir126-test-2-Fw AGAGCCAGGCGCTGGGTCAC, as shown in SEQ ID NO.22 mir126-test-2-Rv

The PCR reaction system is shown in Table 5:

table 5

94°C for 5min; 94°C for 30sec, 62°C for 30sec, 72°C for 30sec, a total of 35 cycles; 72°C for 10min. The PCR amplification product was diluted 10 times, and the second round of PCR was performed using the diluted product as a template. The PCR system is shown in Table 6:

Table 6

94°C for 5min; 94°C for 30sec, 62°C for 30sec, 72°C for 2min, a total of 35 cycles; 72°C for 5min. The PCR amplification products were detected by 1% agarose gel electrophoresis (results are shown in Figure 2 and Figure 4), and the PCR products that were significantly smaller than the untransfected 293T cell sample bands were selected and sent for sequencing ( Shanghai Meiji Biomedical Technology Co., Ltd.) (the results are shown in Figure 3 and Figure 5).

Analysis of the sequencing results by Blast showed that pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A-Puro-sgRNA3 and pSpCas9(BB)-2A-Puro-sgRNA2 and pSpCas9(BB)-2A- The sgRNA plasmid combination composed of Puro-sgRNA5 can completely knock out the miR-126 genome sequence in 293T cells, and the combined deletion of pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A-Puro-sgRNA3 can get positive results More quantity, therefore better efficiency. The knockout verification results of other sgRNA paired combinations were negative, and the miR-126 genome sequence in 293T cells could not be effectively knocked out.

Therefore, pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A-Puro-sgRNA3, pSpCas9(BB)-2A-Puro-sgRNA2 and pSpCas9(BB)-2A-Puro-sgRNA5 were selected as miR -126 sgRNA combinations of full-length gene knockout kit; preferably pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A-Puro-sgRNA3 combinations.

(4) Construct miR-126 knockout 293T cell model and test the knockout efficiency by qPCR

The selected pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A-Puro-sgRNA3 are the sgRNA combination of the miR-126 full-length gene knockout kit, according to the steps of Example 2 (1)— —(3) Obtain the 293T cell line containing miR-126 knockout, count the 293T cells that have been verified to contain miR-126 knockout, dilute to 2/well and inoculate in a 24-well plate, place at 37°C, 5 The culture was carried out in a %CO ₂ incubator for two weeks, during which the growth status of the single clones in the well plate was observed every day. Two weeks later, if the monoclonal growth into a cell cluster in the well plate, take the cells in the cell cluster for PCR verification and sequencing to see if the cell cluster is a 293T monoclonal cell cluster with miR-126 knocked out (see Figure 6 for details). ), the PCR results showed that the band of the 293T monoclonal cell group was significantly smaller than the PCR band of the control 293T cell group, and the PCR product of the 293T monoclonal cell group was sent for sequencing (Shanghai Meiji Biomedical Technology Co., Ltd., the results are shown in Figure 7 ), the sequencing results were verified by Blast comparison, and the miR-126 genome coding sequence was completely knocked out in the 293T monoclonal cell cluster (the monoclonal name was D2). The 126 knockout 293T cell model was successfully constructed.

The successfully constructed 293T knockout miR-126 stable cell clone D2 was expanded and cultured, and RNA (including microRNA) was extracted using the QIAGENmiRNeasy Mini Kit (QIAGEN, Cat. Product No. 218160), the system is shown in Table 7:

Table 7

37°C for 60 minutes; 95°C for 5 minutes. After diluting the inverted cDNA 10 times, use the primers shown in Table 8 to perform qPCR.

Table 8

*Primer sequence (5'--3') *Primer name TCGTACCGTGAGTAATAATGCG, as shown in SEQ ID NO.23 mir126Fw GATTGAATCGAGCACCAGTTAC, as shown in SEQ ID NO.24 common q TTCGTGAAGCGTTCCATATTTT, as shown in SEQ ID NO.25 wxya

The PCR reaction system is shown in Table 9:

Table 9

95°C for 2min; 94°C for 15sec, 60°C for 1min, a total of 40 cycles; at 60°C for 1min, the signal was collected on the SYBR channel of the fluorescent quantitative PCR instrument. After the experiment, use the software that comes with the fluorescent quantitative PCR instrument to analyze the experimental results, and obtain the expression level of miR-126 of 293T monoclonal D2 (239T monoclonal stable strain that has been successfully knocked out) relative to its own U6, and the blank control sample The expression level of miR-126 relative to its own U6. Taking the relative expression of miR-126 in the blank control sample as the standard, it was defined as the expression efficiency of 100%, thus the expression efficiency of miR-126 in the 293T monoclonal D2 was calculated to be 5.37% (the results are shown in Figure 8), It shows that the combination of pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A-Puro-sgRNA3sgRNA can knock out miR-126 in 293T, and the knockout efficiency can reach 94.63%.

Example 3 Construction of miR-126 knockout lung cancer cell model

1) Transfection of lung cancer cells A549 cells

Co-transfect lung cancer cell A549 with pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A-Puro-sgRNA3 using Lipofectamine 2000. After 24 hours of transfection, use the complete medium containing 2 μg/mL puromycin to carry out drug treatment. After three days of drug sieving, the cells were collected.

2) Knockout activity verification

Collect the A549 cells after the drug screening and the A549 cells without transfection, use the ultra-micro sample genotype identification kit (Nanjing Yaoshunyu Biotechnology Co., Ltd., KC-101) for sample processing, and use as shown in Table 10 PCR primers for PCR amplification identification.

Table 10

*Primer sequence (5'--3') *Primer name GAGGGAGGATAGGTGGGTTC, as shown in SEQ ID NO.19 mir126-test-Fw AGGCAGAGCCAGAAGACTCA, as shown in SEQ ID NO.20 mir126-test-Rv GCACTGGAATCTGGGCGGAAGG, as shown in SEQ ID NO.21 mir126-test-2-Fw AGAGCCAGGCGCTGGGTCAC, as shown in SEQ ID NO.22 mir126-test-2-Rv

The PCR reaction system is shown in Table 11:

Table 11

94°C for 5min; 94°C for 30sec, 62°C for 30sec, 72°C for 30sec, a total of 35 cycles; 72°C for 10min. The PCR amplification product was diluted 10 times, and the second round of PCR was performed using the diluted product as a template. The PCR system is shown in Table 12:

Table 12

94°C for 5min; 94°C for 30sec, 62°C for 30sec, 72°C for 2min, a total of 35 cycles; 72°C for 5min. The PCR amplification products were detected by 1% agarose gel electrophoresis, and the PCR products that were significantly smaller than the bands of the untransfected A549 cell samples were selected and sent for sequencing (Shanghai Meiji Biomedical Technology Co., Ltd.). Analysis of the sequencing results using Blast showed that the miR-126 fragment was effectively knocked out in the genome.

3) Isolation of miR-126 knockout lung cancer cell line A549 monoclonal

After counting the lung cancer cell line A549 that has been verified to contain miR-126 knockout, dilute to 2/well and inoculate in a 24-well plate, and place it in a 5% CO ₂ incubator at 37°C for two weeks, during which the wells are observed daily Growth status of single clones in the plate. Two weeks later, if the monoclonal growth into a cell cluster in the well plate, take the cells in the cell cluster for PCR verification and sequencing to see if the cell cluster is an A549 monoclonal cell cluster with miR-126 knocked out (results are shown in Figure 9 for details ), the PCR results showed that the 6 monoclonal bands of A549 were significantly smaller than the PCR bands of the control A549 cells, and the PCR products of monoclonal A2 among the 6 monoclonals were randomly selected and sent for sequencing (Shanghai Meiji Biomedical Technology Co., Ltd., results As shown in Figure 10), the sequencing results were verified by Blast comparison. miR-126 was completely knocked out in the A549 monoclonal A2, and the correct monoclonal cell mass culture and expansion culture will be verified. So far, the miR-126 knockout A549 cells The model build was successful.

Example 4 Construction of an endothelial cell model for miR-126 knockout

1) Human umbilical vein endothelial cells HUVEC cell transfection

Co-transfect human umbilical vein endothelial cells HUVEC with pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A-Puro-sgRNA3 using Lipofectamine 2000. After 24 hours of transfection, use complete medium containing 2 μg/mL puromycin Drug screening was carried out, and after three days of drug screening, the cells were collected.

2) Knockout activity verification

The HUVEC cells after the drug screening and the HUVEC cells without transfection were collected, and the ultra-micro sample genotype identification kit (Nanjing Yaoshunyu Biotechnology Co., Ltd., KC-101) was used for sample processing, and the samples were processed as shown in Table 13. PCR primers for PCR amplification identification.

Table 13

*Primer sequence (5'--3') *Primer name GAGGGAGGATAGGTGGGTTC, as shown in SEQ ID NO.19 mir126-test-Fw AGGCAGAGCCAGAAGACTCA, as shown in SEQ ID NO.20 mir126-test-Rv GCACTGGAATCTGGGCGGAAGG, as shown in SEQ ID NO.21 mir126-test-2-Fw AGAGCCAGGCGCTGGGTCAC, as shown in SEQ ID NO.22 mir126-test-2-Rv

The PCR reaction system is shown in Table 14:

Table 14

94°C for 5min; 94°C for 30sec, 62°C for 30sec, 72°C for 30sec, a total of 35 cycles; 72°C for 10min. The PCR amplification product was diluted 10 times, and the second round of PCR was performed using the diluted product as a template. The PCR system is shown in Table 15:

Table 15

94°C for 5min; 94°C for 30sec, 62°C for 30sec, 72°C for 2min, a total of 35 cycles; 72°C for 5min. The PCR amplification products were detected by 1% agarose gel electrophoresis, and PCR products that were significantly smaller than the bands of untransfected HUVEC cell samples were selected and sent for sequencing (Shanghai Meiji Biomedical Technology Co., Ltd.). Analysis of the sequencing results using Blast showed that the miR-126 fragment was effectively knocked out in the genome.

3) Isolation of miR-126 knockout HUVEC monoclonal

After counting the HUVEC cells that have been verified to contain miR-126 knockout, dilute to 2 cells/well and inoculate in a 24-well plate, place them in a 5% CO ₂ incubator at 37°C for two weeks, and observe the cells in the well plate every day. The growth state of the clone. Two weeks later, if the monoclonal growth into a cell cluster in the well plate, take the cells in the cell cluster for PCR verification and sequencing to see if the cell cluster is a HUVEC monoclonal cell cluster with miR-126 knocked out (see Figure 11 for details). ), the PCR results showed that the band of the monoclonal cell mass of HUVEC was significantly smaller than that of the control HUVEC cells, and the PCR product of the monoclonal cell mass of HUVEC (named monoclonal H1) was sent for sequencing (Shanghai Meiji Biomedical Technology Co., Ltd. , the results are shown in Figure 12), the sequencing results were verified by Blast comparison, miR-126 was completely knocked out in the HUVEC monoclonal cell cluster H1, and the correct monoclonal cell cluster culture will be verified and expanded, so far miR-126 knockout The removed HUVEC cell model was successfully constructed.

Example 5 Knockout of breast cancer cell T47D cell line miR-126

1) Breast cancer cell T47D cell transfection

Co-transfect breast cancer cells T47D with pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A-Puro-sgRNA3 using Lipofectamine 2000. After 24 hours of transfection, use complete medium containing 2 μg/mL puromycin Drug sieving, after three days of drug sieving, the cells were collected.

2) Knockout activity verification

The T47D cells after the drug screening and the T47D cells without transfection were collected, and the ultra-micro sample genotype identification kit (Nanjing Yaoshunyu Biotechnology Co., Ltd., KC-101) was used for sample processing, and the samples were processed as shown in Table 16. The PCR primers were used for PCR amplification identification.

Table 16

*Primer sequence (5'--3') *Primer name GAGGGAGGATAGGTGGGTTC, as shown in SEQ ID NO.19 mir126-test-Fw AGGCAGAGCCAGAAGACTCA, as shown in SEQ ID NO.20 mir126-test-Rv GCACTGGAATCTGGGCGGAAGG, as shown in SEQ ID NO.21 mir126-test-2-Fw AGAGCCAGGCGCTGGGTCAC, as shown in SEQ ID NO.22 mir126-test-2-Rv

The PCR reaction system is shown in Table 17:

Table 17

94°C for 5min; 94°C for 30sec, 62°C for 30sec, 72°C for 30sec, a total of 35 cycles; 72°C for 10min. The PCR amplification product was diluted 10 times, and the second round of PCR was performed using the diluted product as a template. The PCR system is shown in Table 18:

Table 18

94°C for 5min; 94°C for 30sec, 62°C for 30sec, 72°C for 2min, a total of 35 cycles; 72°C for 5min. The PCR amplification product was detected by 1% agarose gel electrophoresis, and the PCR product (as shown in Figure 13) that was obviously smaller than the band of the untransfected T47D cell sample was selected and sent for sequencing (Shanghai Meiji Biotechnology Co., Ltd. Pharmaceutical Technology Co., Ltd). Analysis of the sequencing results (as shown in FIG. 14 ) using Blast showed that the miR-126 fragment was effectively knocked out in the genome.

According to the above experimental results, the present invention determines that sgRNA-1 and sgRNA-3 designed for the miR-126 gene; Cas9 Plasmid for Full-Length Gene Knockout Kit. Experiments have proved that the kit can effectively achieve gene knockout at the cellular level, and can be used in various cell lines including tool cell line 293T, tumor cell lines: lung cancer cell line A549, breast cancer cell line T47D, and endothelial cell line HUVEC, etc. Gene knockout of miR-126. The various cell models of miR-126 gene knockout can be used for poor differentiation of vascular endothelial cells and gene function research in various malignant tumors such as: breast cancer, lung cancer, etc., laying the foundation for subsequent animal modeling and for further research Applied research such as drug development provides convenient tools.

sequence listing

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<120> A miR-126 full-length gene knockout kit based on CRISPR-Cas9 technology and its application

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Claims (9)

1. a kind of miR-126 full-length genes based on CRISPR-Cas9 technologies knock out test kit, it is characterised in that

The test kit is included by plasmid pSpCas9 (BB) -2A-Puro-sgRNA1 and plasmid pSpCas9 (BB) -2A-Puro- The sgRNA plasmid combinations of sgRNA3 compositions；Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1, containing one section of complementation DNA sequence, the DNA sequence can be transcribed into the target of miR-126 full-length gene of the specific recognition sequence as shown in SEQ ID NO.2 The sgRNA of mark sequence, the sgRNA can constitute special identification structure with trRNA, so that guiding Cas9 enzymes specifically shear miR- 126 gene pairss answer sequence；Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA3, the DNA sequence containing one section of complementation should DNA sequence can be transcribed into the target sequence of miR-126 full-length gene of the specific recognition sequence as shown in SEQ ID NO.4 SgRNA, the sgRNA can constitute special identification structure with trRNA, so that guiding Cas9 enzymes specifically shear miR-126 genes Correspondence sequence；

Or, the test kit is included by plasmid pSpCas9 (BB) -2A-Puro-sgRNA2 and plasmid pSpCas9 (BB) -2A- The sgRNA plasmid combinations of Puro-sgRNA5 compositions；Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA2, containing one section mutually The DNA sequence of benefit, the DNA sequence can be transcribed into miR-126 full-length gene of the specific recognition sequence as shown in SEQ ID NO.3 Target sequence sgRNA, the sgRNA can constitute special identification structure with trRNA, so that guiding Cas9 enzymes are specifically sheared MiR-126 gene pairss answer sequence；Plasmid pSpCas9 (the BB) -2A-Puro-sgRNA5, the DNA sequence containing one section of complementation, The DNA sequence can be transcribed into the target sequence of miR-126 full-length gene of the specific recognition sequence as shown in SEQ ID NO.6 SgRNA, the sgRNA can constitute special identification structure with trRNA, so that guiding Cas9 enzymes specifically shear miR-126 genes Correspondence sequence.

2. test kit as claimed in claim 1, it is characterised in that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1 contains Some complementary dna sequences are SEQ ID NO.7 and SEQ ID NO.8.

3. test kit as claimed in claim 1, it is characterised in that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA3 contains Some complementary dna sequences are SEQ ID NO.11 and SEQ ID NO.12.

4. test kit as claimed in claim 1, it is characterised in that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA2 contains Some complementary dna sequences are SEQ ID NO.9 and SEQ ID NO.10.

5. test kit as claimed in claim 1, it is characterised in that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA5 contains Some complementary dna sequences are SEQ ID NO.15 and SEQ ID NO.16.

6. test kit as claimed in claim 1, it is characterised in that plasmid pSpCas9 (the BB) -2A-Puro-sgRNA1 and The sgRNA plasmid combinations of plasmid pSpCas9 (BB) -2A-Puro-sgRNA3 compositions, plasmid pSpCas9 (the BB) -2A- The sgRNA plasmid combinations of Puro-sgRNA2 and plasmid pSpCas9 (BB) -2A-Puro-sgRNA5 compositions are with including following step What rapid method built：

Step one, by target software design preferably a number of miR-126 upstream region of gene and downstream CRISPR-Cas9 targets Mark sequence, it is specific as follows：

Upstream：

sgRNA-1：TAATGTCCCGTCGCCAGCGG, as shown in SEQ ID NO.2；

sgRNA-2：GCCACGCCTCCGCTGGCGAC, as shown in SEQ ID NO.3；

Downstream：

sgRNA-3：TCTCAGCGGCGTTTTCGATG, as shown in SEQ ID NO.4；

sgRNA-4：GAGTAATAATGCGCCGTCCA, as shown in SEQ ID NO.5；

sgRNA-5：TTTCGATGCGGTGCCGTGGA, as shown in SEQ ID NO.6；

Step 2, separately designs sgRNA fragment of the synthesis for upstream and downstream target sequence, and is building up in plasmid vector； The sgRNA fragment sequences are as follows：

sgRNA-1F:CcacgTAATGTCCCGTCGCCAGCGG, as shown in SEQ ID NO.7；

sgRNA-1R:AaacCCGCTGGCGACGGGACATTAc, as shown in SEQ ID NO.8；

sgRNA-2F:CcacGCCACGCCTCCGCTGGCGAC, as shown in SEQ ID NO.9；

sgRNA-2R:Aaac GTCGCCAGCGGAGGCGTGGC, as shown in SEQ ID NO.10；

SgRNA-3F ccacgTCTCAGCGGCGTTTTCGATG, as shown in SEQ ID NO.11；

SgRNA-3R aaacCATCGAAAACGCCGCTGAGAC, as shown in SEQ ID NO.12；

SgRNA-4F ccacGAGTAATAATGCGCCGTCCA, as shown in SEQ ID NO.13；

SgRNA-4R aaacTGGACGGCGCATTATTACTC, as shown in SEQ ID NO.14；

SgRNA-5F ccacgTTTCGATGCGGTGCCGTGGA, as shown in SEQ ID NO.15；

SgRNA-5R aaacTCCACGGCACCGCATCGAAAc, as shown in SEQ ID NO.16；

Step 3, by transfecting 293T cell strains, sgRNA and trRNA constitutes special identification structure, so as to guide Cas9 enzymes special The corresponding sequence at strange land shearing miR-126 genes two ends, continues medicine sieve, obtains the cell strain of miR-126 full-length genes knockout；

Step 4, by extracting cell DNA, PCR amplifications, sequencing is determined and verifies, obtains the two groups of miR-126 bases for optimizing SgRNA plasmid combinations because knocking out CRISPR-Cas9, i.e., described plasmid pSpCas9 (BB) -2A-Puro-sgRNA1 and plasmid The sgRNA plasmid combinations of pSpCas9 (BB) -2A-Puro-sgRNA3 compositions, and the plasmid pSpCas9 (BB) -2A- The sgRNA plasmid combinations of Puro-sgRNA2 and plasmid pSpCas9 (BB) -2A-Puro-sgRNA5 compositions.

7. test kit as claimed in claim 6, it is characterised in that described to be building up in plasmid vector in step 2, concrete bag Include following steps：

(1) with pSpCas9 (BB) -2A-Puro plasmids as initial vector, single endonuclease digestion is carried out with Bbs I, linearized vector is carried out Purification；

(2) synthetic sgRNA fragments are diluted and is annealed；

(3) linearizing pSpCas9 (BB) -2A-Puro is connected with the sgRNA fragments after annealing using T4DNA ligases, will Whole connection products add the conversion of bacillus coli DH 5 alpha competent cell；

(4) single bacterium colony that the flat board of DH5 α competent cells bacterium solution coating after conversion grows is identified using bacterium colony PCR methods, Obtain expression plasmid.

8. application of the test kit as described in any one of claim 1-7 in cell line miR-126 gene knockout product is prepared.

9. application as claimed in claim 8, it is characterised in that the cell line includes vehicles cells system 293T, tumor cell System and endotheliocyte HUVEC systems.