CN104212836A - Method for knocking out mir-505 from mammal cell line - Google Patents

Abstract

The present invention relates to a method for knocking out mir-505 in a mammalian cell line. The nucleotide single strand of sgRNA is synthesized in vitro, and then processed to obtain an insert fragment, and then the sgRNA is inserted into 41824 by homologous recombination or T4 connection In the vector or 42230 vector, transform into competent cells of Escherichia coli, and then perform colony PCR detection and sequencing confirmation on the vector to obtain the expression vector, and form a heterologous hybrid double strand by denaturing the PCR product and annealing, using T7E1 The enzyme digestion test was used to determine the cutting efficiency of the mir-505 gene by the CRISPR-Cas9 system. The invention provides a reference for how to select sgRNA expression vectors.

Description

A method for knocking out mir-505 in mammalian cell lines

technical field

The invention belongs to the field of gene knockout methods, in particular to a method for knocking out mir-505 in mammalian cell lines.

Background technique

In 1987, the Japanese research group discovered tandem spaced repeats near the alkaline phosphatase gene of E.coli K12. In subsequent studies, it was found that such spaced repeats widely exist in the genomes of bacteria and archaea. In 2002, , officially named by scientists as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

CRISPR has been found to be an immune system of bacteria and archaea, which consists of a series of short conserved repeat sequences separated by unique DNA sequences of similar size, a unique DNA sequence called a spacer, and these spacers Usually derived from phage or plasmid DNA. CRISPR column and Cas (CRISPR-associated) gene form CRISPR-Cas adaptive immune system ^[5-7] . The function of the CRISPR/Cas system is as follows: the invading nucleic acid fragments are incorporated into the host genome as spacers, and then these spacers are used as templates to generate small RNA molecules (crRNA), which can combine with Cas proteins to form effector complexes It can silence the exogenous nucleic acid in the next round of infection.

The Type II system requires only the Cas9 protein (formerly known as Cas5 or Csn1) for DNA interference. The steps of the Type II system for double-strand break of the target gene: (i) pre-crRNA and tracrRNA are transcribed from the CRISPR site; (ii) tracrRNA and the direct repeat sequence in the pre-crRNA hybridize to form a double strand, and combine with Combined with Cas9, pre-crRNA forms mature crRNA through the action of RNase III and an unknown nuclease. The mature crRNA contains a short spacer sequence; (iii) mature crRNA: tracrRNA hybrid double strand guides the Cas9 protein to the target DNA site, and the DNA target site needs to contain protospacer and necessary PAM (protospacer adjacent motif), this process It is accomplished through the formation of hybrid double strands between the spacer sequence of crRNA and protospacer DNA; (iv) Cas9 protein mediates cleavage upstream of the PAM in the target DNA, and double-strand breaks in the protospacer (Figure 1).

The CRISPR/Cas9 system has become the hottest gene editing tool in the past year, and it is used in gene knockout research in mice, rats, macaques and other mammals.

In 2010, Verduci L et al. found that miR-505 could regulate the proliferation and senescence/apoptosis of mouse embryonic fibroblasts by acting on its target ASF/SF2 (alternative splicing factor). Karni R et al. found that in many cells Transfection of ASF/SF2 in medium can activate part of the mTOR signaling pathway, but the specific way in which ASF participates in the regulation of mTOR is still unclear. Yamamoto Y et al. found that miR-505 is a new tumor suppressor miRNA when studying tumor multidrug resistance, and it is negatively related to the protein Akt3, which belongs to the same gene family as AKT on the mTOR pathway. Therefore, miR-505 is very likely to exert its biological function by regulating the mTOR signaling pathway, and the mTOR pathway is currently a hot spot in molecular biology research. It can integrate intracellular and intercellular signals to regulate cell metabolism, growth, and proliferation. As a central regulator of processes such as cancer and survival, changes in the mTOR pathway are associated with a variety of diseases.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for knocking out mir-505 in mammalian cell lines. The method of the present invention provides a reference for how to select sgRNA expression vectors.

A method of knocking out mir-505 in a mammalian cell line of the present invention, comprising:

(1) Design sgRNA for the mir-505 gene, then synthesize the nucleotide single strand of the sgRNA in vitro, anneal, and extend it into a double-stranded DNA fragment. The 41824 vector is digested with Afl II to obtain the 41824 linear vector, and then the 41824 linear vector and double-stranded DNA fragments were inserted using the method of homologous recombination, and then transformed into E. coli competent cells DH5α, spread on 1% ampicillin-resistant agarose LB plates until colonies grew, and then performed PCR on single colonies Identify, sequence, and obtain the sgRNA expression vector, wherein the nucleotide single strand of the sgRNA is:

sgRNA3-F:5'-TTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTAAATTGATGCACCCAGTG; (such as SEQ ID NO:1)

sgRNA3-R:5'-ACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACCACTGGGTGCATCAATTTAC; (such as SEQ ID NO:2)

Or synthesize the nucleotide single strand of sgRNA in vitro, anneal to a double-stranded DNA fragment, and digest the 42230 vector with Bbs I to obtain a 42230 linear vector, and then connect the 42230 linear vector and the double-stranded DNA fragment by T4DNA ligase for fragment insertion. Then transformed into competent cells DH5α of Escherichia coli, spread on 1% ampicillin-resistant agarose LB plate until colonies grow, and sequenced to obtain sgRNA/Cas9 co-expression vector; wherein the nucleotide single strand of sgRNA synthesized in vitro is :

sgRNA3-F:5'-CACCGTAAATTGATGCACCCAGTG-3'; (SEQ ID NO:3)

sgRNA3-R:5'-AAACCACTGGGTGCATCAATTTAC-3'; (SEQ ID NO:4)

sgRNA4-F:5'-CACCGCAGAAACATCAATACTTCC-3'; (SEQ ID NO:5)

sgRNA4-R:5'-AAACGGAAGTATTGATGTTTCTGC-3'; (SEQ ID NO:6)

(2) Transfect the above-mentioned expression vector into mammalian cells, and after incubation for 48-72 hours, extract the genomic DNA of the cells, and then use PCR to amplify the fragment including the mir-505 gene, and then denature and anneal the PCR product The heterologous hybrid duplex is formed by T7E1 enzyme digestion and 2% agarose gel electrophoresis to obtain the shearing efficiency of each cell group.

The nucleotide single strand of the sgRNA synthesized in the step (1) is annealed and extended, wherein the PCR system: add ddH2O 10ul, GT Buffer, 1.5ul, dNTP 1.5ul, positive and negative single strand (10P) 1.5ul, BSA 0.2 ul, taq enzyme 1ul. PCR program: 95°C for 2min, 5 cycles of (95°C for 30sec, 56°C for 30sec, 72°C for 1min), 72°C for 10min.

PCR identification in the step (1), the primers identified by PCR in the sequencing are: 41824-AflII-L: 5'TTTCTTGGGTAGTTTGCAGTTTT (such as SEQ ID NO:7), 41824-AflII-R: 5'TTGTACAAGAAAGCTGGGTCT (such as SEQ ID NO :8); Bidirectional sequencing primers are: 41824-AflII-L: 5'TTTCTTGGGTAGTTTGCAGTTTT (such as SEQ ID NO:9), 41824-AflII-R: 5'TTGTACAAGAAAGCTGGGTCT (such as SEQ ID NO:10).

The PCR system in the colony PCR identification in the step (1): add ddH2O 9.3ul, GT Buffer 1.5ul, dNTP 1.5ul, Primer (2P) 1.5ul, BSA 0.2ul, taq enzyme 1ul, and finally pick a single colony as a template ; PCR program: 95°C for 2min, (95°C for 30sec, 56°C for 30sec, 72°C for 1min30sec) 40 cycles, 72°C for 10min.

To obtain positive clone candidates, pick a single colony and shake the bacteria to obtain the bacterial liquid, take 1ml of the bacterial liquid and 20ul 10P upstream and downstream primers (upstream primer, downstream primer 10P each), and perform bidirectional sequencing.

In the step (1), anneal to double-stranded DNA; system: add ddH2O 8.5ul, positive and negative single-stranded (10P) 5ul, GT Buffer 1.5ul. Procedure: After 4 minutes at 95°C, place at room temperature for 10 minutes.

In the step (1), the one-way sequencing primer is: 42230-BbsI-R: 5'CGACTCGGTGCCACTTTTT (such as SEQ ID NO: 11), randomly select a single colony and shake the bacteria to obtain the bacterial liquid, take 1ml of the bacterial liquid and 20ul of 10P downstream primers, Perform one-way sequencing. The mammalian cells in the step (2) are HEK293A cells.

The incubation condition in the step (2) is 37° C., incubating in an incubator with 5% CO ₂ .

In the step (2), the fragment containing the mir-505 gene is amplified by PCR: PCR system: add ddH ₂ O 9.5ul, 2X hotstart MIX 12.5ul, Primer (10P) 1ul, Template 2ul; PCR program : 94°C for 3min, 35 cycles of (94°C for 30sec, 63.2°C for 30sec, 72°C for 1min), 72°C for 5min.

In the described step (2), the amplification primer is:

mir-505-L: AGTGTCAGGCCTGCTATGTT; (such as SEQ ID NO: 12)

mir-505-R:CTCCTGGCTTCTACTCCCTG. (eg SEQ ID NO:13)

In the step (2), the PCR product obtained by PCR amplification is recovered and quantified by using a kit, and 500ng of the product is taken for denaturation and annealing to form a heterologous hybrid double strand; reaction system: 500ng of PCR product, T7E1Buffer 1.1ul, with ddH ₂ O replenish the system to 11ul. Reaction program: 95°C for 10 min, (95°C for 30 sec, a total of 70 cycles, the temperature drops by 1°C in each cycle), 25°C for 1 min. After obtaining the heterologous hybrid duplex, add 0.5ul T7E1 enzyme to the above system, react at 37°C for 30min, and carry out enzyme digestion.

The invention establishes a method for knocking out the mir-505 gene in a mammalian cell line. Design different sgRNAs for the mir-505 gene, and then insert the sgRNAs into the sgRNA expression vector (plasmid: Addgene 41824), or insert the designed sgRNAs into sgRNA/Cas9 co-expression in order to improve the co-delivery of sgRNA and Cas9 protein Vector (plasmid: Addgene 42230), so as to achieve the purpose of target cleavage. And through the detection of cutting efficiency, it is known whether the co-expression of the same sgRNA and Cas9 protein affects the cutting efficiency of the target site, and whether different sgRNAs designed for the same gene have different cutting efficiencies for the gene.

The present invention first designs two different sgRNAs for the mir-505 gene, one of which targets the upstream of the mir505-5P mature body (sgRNA3), and the other target is located in the mir505-5P mature body (sgRNA4). When constructing the sgRNA expression vector, use the in vitro synthesis method to synthesize two partially complementary DNA single strands, and then anneal and extend them to form a long DNA double-stranded fragment containing the sgRNA, and the fragment is two The two ends were homologous to the sequences at both ends of the Afl II restriction site of the 41824 vector. The 41824 vector was digested by Afl II and linearized, and then the DNA fragment was inserted into the Afl II restriction site of the 41824 vector by means of homologous recombination. When constructing the co-expression vector of sgRNA and Cas9 protein, two single-stranded nucleotides are synthesized in vitro, and then annealed in vitro to make the two single-stranded nucleotides form double-stranded DNA through base complementarity, which is the specific design sgRNA with specific cohesive ends at both ends. The 42230 vector was digested with Bbs I, and the linearized vector was purified; the purified linearized vector was linked with two different double-stranded sgRNAs at 14°C overnight. All the constructed vectors were transformed into Escherichia coli competent cells DH5α, the plasmids were extracted, and the sgRNA expression vector and sgRNA/Cas9 co-expression vector were obtained after colony PCR identification and sequencing to confirm the correct insertion of sgRNA. The vectors were transfected into mammalian cells, and the T7E1 enzyme digestion test was used to verify that the mir-505 genome was cut.

The constructed sgRNA expression vector targeting mir-505 and the Cas9 expression vector were co-transfected into mammalian cells or the sgRNA/Cas9 co-expression vector was transfected into mammalian cells, and after 72 hours, the genomic DNA of the cells was extracted. A fragment of about 1 kb in size including the mir505 gene was amplified by PCR method, and then the PCR product was denatured and then annealed to form a heterologous hybrid duplex, and the T7E1 enzyme digestion test was used to determine the effect of the CRISPR-Cas9 system on mir-505 Gene splicing efficiency.

Beneficial effect

The present invention constructs a vector capable of simultaneously expressing Cas9 protein and sgRNA in mammalian cells, and designs sgRNAs with different GC% for the same mir-505 gene, and inserts the sgRNA into the sgRNA/Cas9 co-expression vector for use in To study whether different sgRNAs have different cleavage efficiencies for the same gene; at the same time, sgRNAs are inserted into sgRNA expression vectors to study whether sgRNAs and Cas9 have different cleavage efficiencies for target sites;

In the present invention, a synthetic sgRNA fragment is inserted into the Bbs I restriction site of the 42230 carrier, and a positive clone is obtained by sequencing; at the same time, a synthetic sgRNA fragment is inserted into the Afl II restriction site of the 41824 carrier, and a positive clone is obtained through colony PCR and sequencing clone. The obtained co-expression vectors inserted with different sgRNAs are transfected into mammalian cells or co-transfected with sgRNA expression vectors and Cas9 expression vectors, and then the genomic DNA of the transfected cells is extracted and amplified by PCR The fragment including the mir505 gene was obtained, and the T7E1 enzyme digestion test can confirm that sgRNAs with different GC% do have an impact on the shearing efficiency of the same gene, and a low GC% will correspondingly reduce the shearing efficiency of its target, which will be used for future research. Design and selection of sgRNA provide certain reference. And because the sgRNA/Cas9 co-expression vector improves the co-delivery of sgRNA and Cas9 protein, it can indeed improve the shearing efficiency of the target, providing a reference for how to choose the sgRNA expression vector.

Description of drawings

Figure 1 is a schematic diagram of Type II CRISPR-mediated DNA double-strand breaks;

Fig. 2 is the schematic diagram of constructing mir-505sgRNA expression vector;

Figure 3 is a schematic diagram of constructing mir-505sgRNA/Cas9 co-expression vector;

Figure 4 is the electrophoresis of the sgRNA insert fragment of 41824, where lane 1: marker, lane 2: DNA fragment after annealing extension, lane 3: single-stranded nucleotide without annealing extension;

Figure 5 shows the electrophoresis of sgRNA insert fragments formed by 42230 using different buffer systems, where lane 1: marker, lane 2: single-stranded nucleic acid annealed in H2O, lane 3: single-stranded nucleic acid annealed in enzyme digestion buffer, lane 4: single-stranded nucleic acid Annealed in PCR buffer, lane 5: single-stranded nucleic acid is not annealed;

Figure 6 shows the PCR structure of the 41824-mir-505-sgRNA3 vector colony, where lane 1: marker, lane 2-16: monoclonal, lane 17: negative, lane 18: marker;

Figure 7 shows the structure of the T7E1 digestion test, in which lane 1: marker, lane 2: 375ng42230 vector, 125ng41824-mir-505-sgRNA3 vector; lane 3: 500ng42230 vector, 200ng41824-mir-505-sgRNA3 vector; lane 4: 750ng42230 vector , 250ng41824-mir-505-sgRNA3 vector, lane 5: wild type; lane 6: 375ng 42230-mir-505-sgRNA3, lane 7: 500ng 42230-mir-505-sgRNA3, lane: 750ng42230-mir-505-sgRNA3, Lane 9: 375ng 42230-mir-505-sgRNA4, Lane 10: 500ng 42230-mir-505-sgRNA4, Lane 11: 759ng 42230-mir-505-sgRNA4.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

(1) For the mir-505 gene, use the online sgRNA design software to design sgRNA

sgRNA3: GC%: 52%;

sgRNA4: GC%: 43%

(2) In vitro synthesis of single-stranded nucleotide chains and formation of DNA double-stranded fragments

A. mir-505sgRNA3 insert in 41824

sgRNA3-F:5'-TTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTAAATTGATGCACCCAGTG

sgRNA3-R:5'-ACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACCACTGGGTGCATCAATTTAC

Using the PCR system, the two single-stranded nucleotides were annealed and extended into a double-stranded DNA fragment with a sequence length of 98 bp, which was identified by 2% agarose gel electrophoresis ( FIG. 4 ). PCR system: add ddH2O 10ul, GT Buffer, 1.5ul, dNTP 1.5ul, positive and negative single-stranded (10P) 1.5ul, BSA 0.2ul, taq enzyme 1ul. PCR program: 95°C for 2min, 5 cycles of (95°C for 30sec, 56°C for 30sec, 72°C for 1min), 72°C for 10min.

mir-505sgRNA3 and mir-505sgRNA4 inserts in B.42230

sgRNA3-F:5'-CACCGTAAATTGATGCACCCAGTG-3'

sgRNA3-R:5'-AAACCACTGGGTGCATCAATTTAC-3'

sgRNA4-F:5'-CACCGCAGAAACATCAATACTTCC-3'

sgRNA4-R:5'-AAACGGAAGTATTGATGTTTCTGC-3'

Using different buffers, the two single-stranded nucleotides were annealed into double-stranded DNA fragments, which were identified by 2% agarose gel electrophoresis ( FIG. 5 ). System 1: add H2O 10ul, positive and negative single chain (10P) 5ul; system 2: H2O 8.5ul, positive and negative single chain (10P) 5ul, GT Buffer 1.5ul; system 3: H2O 8.5ul, positive and negative single chain (10P ) 5ul, T7E1Buffer 1.5ul. Procedure: After 4 minutes at 95°C, place at room temperature for 10 minutes.

(3) Construction of 41824-mir-505-sgRNA3 and 42230-mir505-sgRNA vectors

A. The 41824 vector was digested with Afl II, and the linearized vector 41824 and double-stranded sgRNA3 were inserted into fragments by homologous recombination, and transformed into E. coli competent cells DH5α, and coated with 1% ampicillin-resistant Agarose LB plate until colonies grow, use the following pair of primers for PCR identification of single colonies, 41824-AflII-L: 5'TTTCTTGGGTAGTTTGCAGTTTT, 41824-AflII-R: 5'TTGTACAAGAAAGCTGGGTCT

2% agarose gel electrophoresis identification, short fragments indicate that there is no mir-505-sgRNA3 fragment inserted, and long fragments indicate that mir-505-sgRNA3 is inserted. PCR system for colony PCR identification: add ddH2O 9.3ul, GT Buffer 1.5ul, dNTP 1.5ul, Primer (2P) 1.5ul, BSA 0.2ul, taq enzyme 1ul, and finally pick a single colony as a template. PCR program: 95°C for 2min, 40 cycles (95°C for 30sec, 56°C for 30sec, 72°C for 1min30sec), 72°C for 10min.

In order to ensure that the bases of the inserted fragments are completely correct, the positive clones screened by colony PCR were then sequenced and identified. Bidirectional sequencing primers: 41824-AflII-L: 5'TTTCTTGGGTAGTTTGCAGTTTT, 41824-AflII-R: 5'TTGTACAAGAAAGCTGGGTCT. To obtain candidate positive clones, pick a single colony and shake to obtain the bacterial liquid, take 1ml of the bacterial liquid and 20ul 10P upstream and downstream primers (10P for the upstream primer and 10P for the downstream primer), and submit it to the biological company for commercial bidirectional sequencing.

B. The 42230 vector was digested with Bbs I, the linearized vector 42230 and the annealed sgRNA3 and sgRNA4 were ligated by T4 DNA ligase for fragment insertion, and transformed into Escherichia coli competent cells DH5α, coated with 1% ampicillin Sexual agarose LB plates until colonies grow. Since the insert fragments were too small to be distinguished by colony PCR, positive clones were identified by sequencing. Unidirectional sequencing primer: 42230-BbsI-R: 5’CGACTCGGTGCCACTTTT

(4) Transfection of HEK293A cells with 41824-mir-505-sgRNA and 42230-mir-505-sgRNA

Before the experiment, spread HEK293A cells in a 24-well plate at a density of 1.5×10 ⁵ cells/well to ensure that the cells in each well are in a good growth state and have a similar density. When the cells are monolayer and in mid-logarithmic phase, the confluence of the cells is 60%. Transfection was performed at -70%. 41824-mir-505-sgRNA and 42230 vectors co-transfected HEK293A cells with different concentration gradients, and 42230-mir-505-sgRNA vectors also transfected HEK293A cells with different concentration gradients. After transfection, the cytoplasm was incubated for 72 hours at 37°C in a 5% CO ₂ incubator.

(5) CRISPR-Cas9 can be used to cut the mir-505 gene position

Use the animal genomic DNA rapid extraction kit to extract the genomic DNA of the cells, use the PCR method to amplify the long fragment including the mir-505 gene, and amplify the primers mir-505-L:AGTGTCAGGCCTGCTATGTT,mir-505-R:CTCCTGGCTTCTACTCCCTG . After purification of the amplified products, 500 ng of the purified PCR products were taken, and heterologous hybrid double strands were formed by denaturing the products and then annealing and renaturation. The shearing efficiency of each cell population was obtained by cutting the heterologous hybrid duplex with T7E1 enzyme and running 2% agarose gel electrophoresis.

A fragment containing the mir-505 gene was amplified by PCR. PCR system: add ddH ₂ O 9.5ul, 2X hotstart MIX 12.5ul, Primer (10P) 1ul, Template 2ul. PCR program: 94°C for 3min, (94°C for 30sec, 63.2°C for 30sec, 72°C for 1min) 35 cycles, 72°C for 5min.

The PCR product obtained by PCR amplification was recovered and quantified using a kit. Take 500ng of the product for denaturation and annealing to form a heterologous hybridization duplex reaction system: PCR product 500ng, T7E1Buffer 1.1ul, supplement the system with ddH ₂ O to 11ul. Reaction program: 95°C for 10 min, (95°C for 30 sec, a total of 70 cycles, the temperature drops by 1°C in each cycle), 25°C for 1 min. After obtaining the heterologous hybrid duplex, add 0.5ul T7E1 enzyme to the above system, react at 37°C for 30min, and carry out enzyme digestion.

The results are shown in Figure 7, 1#-3# and 5#-7# use the same GC% sgRNA, since 5#-7# is sgRNA and Cas9 protein are co-expressed, so to a certain extent, it improves its target cutting efficiency. 5#-7# and 8#-10# use sgRNA with different GC%, and both sgRNA and Cas9 protein are co-expressed. As a result, the GC% of 8#-10# sgRNA is 43%, which is relatively low, so It also cuts less efficiently at the target site.

Therefore, when using the CRISPR-Cas9 system for microRNA knockout, the GC% should be about 50% when selecting sgRNA, and improving the co-delivery of sgRNA and Cas9 protein can improve its gene knockout efficiency to a certain extent.

Claims (11)

1. in mammal cell line, knock out a method of mir-505, comprising:

(1) for mir-505 gene design sgRNA, then the nucleotide single-chain of external synthesis sgRNA, anneal, extend to double chain DNA fragment, 41824 carrier Afl II carry out enzyme and cut, obtain 41824 linear carriers, then 41824 linear carriers and double chain DNA fragment utilize the method for homologous recombination to carry out fragment insertion, then colibacillary competent cell DH5 α is converted into, be applied to the agarose LB flat board of 1% ammonia benzyl resistance to growing bacterium colony, then PCR qualification is carried out to single bacterium colony, order-checking, obtain sgRNA expression vector, wherein the nucleotide single-chain of sgRNA is:

sgRNA3-F:5’-TTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTAAATTGATGCACCCAGTG；

sgRNA3-R:5’-ACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACCACTGGGTGCATCAATTTAC；

Or the nucleotide single-chain of external synthesis sgRNA, be annealed into double chain DNA fragment, 42230 carrier Bbs I carry out enzyme and cut, obtain 42230 linear carriers, then 42230 linear carriers and double chain DNA fragment are connected by T4DNA ligase enzyme and carry out fragment insertion, being then converted into colibacillary competent cell DH5 α, being applied to the agarose LB flat board of 1% ammonia benzyl resistance to growing bacterium colony, order-checking, obtains sgRNA/Cas9 co-expression carrier; Wherein the nucleotide single-chain of external synthesis sgRNA is:

sgRNA3-F:5’-CACCGTAAATTGATGCACCCAGTG-3’；

sgRNA3-R:5’-AAACCACTGGGTGCATCAATTTAC-3’；

sgRNA4-F:5’-CACCGCAGAAACATCAATACTTCC-3’；

sgRNA4-R:5’-AAACGGAAGTATTGATGTTTCTGC-3’；

(2) by above-mentioned expression vector transfection mammalian cell, after hatching 48-72h, extract cell genomic dna, then pcr amplification is utilized to go out to comprise the fragment of mir-505 gene, then by PCR primer is carried out sex change, the mode of annealing forms Heterologous Hybridization double-strand, Heterologous Hybridization double-strand is cut by T7E1 enzyme, and 2% agarose gel electrophoresis, obtain the shear efficiency that each cell mass occurs.

2. a kind of method knocking out mir-505 in mammal cell line according to claim 1, it is characterized in that: in described step (1), the nucleotide single-chain of synthesis sgRNA is annealed, extend, wherein PCR system: add ddH2O 10ul, GT Buffer, 1.5ul, dNTP 1.5ul, positive and negative strand (10P) 1.5ul, BSA 0.2ul, taq enzyme 1ul; PCR program: 95 DEG C of 2min, then 95 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 1min are 5 circulations, 72 DEG C of 10min.

3. a kind of method knocking out mir-505 in mammal cell line according to claim 1, it is characterized in that: PCR qualification in described step (1), the primer that in order-checking, PCR identifies is: 41824-AflII-L:5 ' TTTCTTGGGTAGTTTGCAGTTTT, 41824-AflII-R:5 ' TTGTACAAGAAAGCTGGGTCT; Sequencing primer is: 41824-AflII-L:5 ' TTTCTTGGGTAGTTTGCAGTTTT, 41824-AflII-R:5 ' TTGTACAAGAAAGCTGGGTCT.

4. a kind of method knocking out mir-505 in mammal cell line according to claim 1, it is characterized in that: in described step (1), bacterium colony PCR identifies, in order-checking, PCR system: add ddH2O 9.3ul, GT Buffer 1.5ul, dNTP1.5ul, Primer (2P) 1.5ul, BSA 0.2ul, taq enzyme 1ul, single bacterium colony is as template; PCR program: 95 DEG C of 2min, then 95 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 1min30sec, 40 circulations, 72 DEG C of 10min;

Obtain positive colony candidate, choose single bacterium colony and shake bacterium and obtain bacterium liquid, get 1ml bacterium liquid and 20ul 10P upstream and downstream primer, upstream primer, each 10P of downstream primer, carries out two-way order-checking.

5. a kind of method knocking out mir-505 in mammal cell line according to claim 1, it is characterized in that: described step is annealed into double-stranded DNA in (1), system: add ddH2O 8.5ul, positive and negative strand (10P) 5ul, GT Buffer 1.5ul; Program: after 95 DEG C of 4min, room temperature places 10min.

6. a kind of method knocking out mir-505 in mammal cell line according to claim 1, it is characterized in that: in described step (1), sequencing primer is: 42230-BbsI-R:5 ' CGACTCGGTGCCACTTTT, picking individual colonies shakes bacterium and obtains bacterium liquid, get 1ml bacterium liquid and 20ul 10P downstream primer, carry out unidirectional order-checking.

7. a kind of method knocking out mir-505 in mammal cell line according to claim 1, is characterized in that: in described step (2), mammalian cell is HEK293A cell.

8. a kind of method knocking out mir-505 in mammal cell line according to claim 1, is characterized in that: in described step (2), incubation conditions is 37 DEG C, 5%CO ₂incubator in hatch.

9. a kind of method knocking out mir-505 in mammal cell line according to claim 1, is characterized in that: in described step (2) with pcr amplification go out to comprise mir-505 gene fragment, PCR system: add ddH ₂o 9.5ul, 2X hotstart MIX 12.5ul, Primer (10P) 1ul, Template 2ul; PCR program: 94 DEG C of 3min, then 94 DEG C of 30sec, 63.2 DEG C of 30sec, 72 DEG C of 1min, 35 circulations, 72 DEG C of 5min.

10. a kind of method knocking out mir-505 in mammal cell line according to claim 1, is characterized in that: in described step (2), amplimer is:

mir-505-L:AGTGTCAGGCCTGCTATGTT；

mir-505-R:CTCCTGGCTTCTACTCCCTG。

11. a kind of methods knocking out mir-505 in mammal cell line according to claim 1, it is characterized in that: the PCR primer that pcr amplification obtains by described step (2) utilizes test kit to carry out product recovery, quantitatively, get 500ng product and carry out sex change annealing formation Heterologous Hybridization double-strand, reaction system: PCR primer 500ng, T7E1Buffer 1.1ul, uses ddH ₂o supplements system to 11ul; Response procedures: 95 DEG C of 10min, then 95 DEG C of 30sec, totally 70 circulations, each circulating temperature falls 1 DEG C, 25 DEG C of 1min, after obtaining Heterologous Hybridization double-strand, adds 0.5ul T7E1 enzyme in above-mentioned system, and 37 DEG C of reaction 30min, carry out enzyme and cut.