CN107604003A - One kind knocks out kit and its application based on linearisation CRISPR CAS9 lentiviral vector genomes - Google Patents

Abstract

The invention discloses a gene knockout kit based on a CRISPR-CAS9 lentiviral vector, which includes a linearized CRISPR-CAS9 lentiviral vector, corresponding T4 ligase and competent cells, and is characterized in that the The linearized CRISPR-CAS9 lentiviral vector is composed of an endonuclease-treated circular vector, the T4 ligase is an optimized T4 ligase, and the competent cells are Stbl3 competent with the property of inhibiting recombination. The kit can directly construct the CRISPR‑CAS9 lentiviral vector, which is more convenient to operate than domestic and foreign commercial kits, and has a faster construction and recombination speed, which can effectively improve the construction efficiency of the current CRISPR‑CAS9-based lentiviral vector and reduce gene Knockout fee.

Description

A linearized CRISPR-CAS9 lentiviral vector gene knockout kit and its application

technical field

The invention relates to the field of biology, in particular to a preparation for research, in particular to a gene knockout kit based on a CRISPR-CAS9 lentiviral vector.

Background technique

Gene modification is an important issue in the current biological field. In recent years, the rapid development of genome editing technology has brought a new era to biological research. Different from traditional gene cloning technology, genome editing technology can directly perform DNA sequence knockout, insertion, site-directed mutation, and combined editing on the genome to realize systematic research on gene function and regulatory elements, and has great potential in industrial bioengineering. Broad application prospects. In the early days, genome editing technology mainly used targeting technology mediated by homologous recombination, but its application was greatly limited due to its low efficiency. To solve this problem, a series of artificial endonuclease-mediated genome editing technologies have been developed, which can form DNA double-strand breaks at specific positions in the genome, with the help of the cell's own repair system such as non-homologous end joining or homologous end-joining. source recombination, enabling efficient site-directed genome editing in different organisms and cell types. Currently, four different artificial endonucleases have been applied to genome editing: meganuclease technology, zinc finger endonucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). Cas9, ZFN and TALEN can recognize specific DNA sequences on the genome through protein-DNA interactions with RNA-targeting DNA endonucleases. Megakaryozyme technology has an endonuclease domain and a DNA binding domain, while ZFN and TALEN have DNA binding domains that can recognize 3 and 1 nucleotides respectively, and need to be formed with the endonuclease domain of the endonuclease FokI After the fusion protein, cleavage at a specific site in the genome is accomplished. However, these technologies have their own shortcomings. For example, there is no clear specificity between the amino acid residues of meganuclease technology and the DNA target sequence; the DNA binding domain of ZFN will be affected by the upstream and downstream of the DNA target sequence, and different fusion proteins need to be constructed. To target different DNA sequences, the construction operation is cumbersome, the cost is high, and the risk of off-target is high. RNA-targeting endonuclease CRISPR—Cas9-mediated genome editing technology uses a short guide RNA (guideRNA) to identify a specific DNA sequence, and Cas9 can be positioned to a new DNA only by changing the guide RNA sequence sequence. This technique has been applied to a variety of organisms, including humans, mice, rats, zebrafish, C. elegans, plants, and bacteria. Its technology has also been continuously expanded. For example, multiple guide RNA sequences can be used to edit multiple different sites on the genome at the same time; its DNA binding domain can be fused with different transcriptional regulatory proteins to achieve transcriptional activation or inhibition of specific genes. ; The dynamic control of the genome can be achieved by fusing the regulatory protein module for a specific sequence with the modification enzyme of the genome or epigenome.

Lentivirus is currently the most widely used technology in the field of transfection. By constructing a lentiviral packaging plasmid, the target gene can be efficiently integrated into the chromosome of the cell, achieving efficient transfection of difficult-to-transfect cells and reducing the cost of expensive transfection reagents. Effect.

CRISPR-Cas9-mediated genome editing technology has become a research hotspot due to its simple operation and high specificity, but there is still a lack of commercialized kits for the operation of gene editing technology. Therefore, the research and development is accurate, reliable, simple and practical, suitable for various The CRISPR-CAS9 lentiviral vector gene knockout kit operated by researchers is an urgent need at present. The present invention combines the high-efficiency transfection characteristics of lentiviral vectors and the high-efficiency gene editing characteristics of CRISPR-CAS9 technology to solve important problems in this field. problem.

Contents of the invention

The technical problem to be solved by the present invention is to control the gene knockout efficiency of the lentiviral vector and reduce the difference of gene knockout.

The technical scheme that the present invention solves the above problems is:

A gene knockout kit based on CRISPR-CAS9 lentiviral vector, the kit includes linearized CRISPR-CAS9 lentiviral vector, T4 ligase and T4 ligase buffer, highly transformable Stbl3 competent cells, characterized in that :

1. The linearized CRISPR-CAS9 lentiviral vector is obtained by the CRISPR-CAS9 lentiviral vector through an endonuclease;

2. The ligase is T4 ligase;

3. The competent cells are highly transformable Stbl3 competent cells prepared by the calcium chloride method.

In the kit of the present invention, the linearized CRISPR-CAS9 lentiviral vectors include but not limited to lentiviral vectors, and can also be used for common CRISPR-CAS9 vectors; T4 ligase is commonly used or commercial T4 ligase; Stbl3 competent Cells were prepared by the calcium chloride method.

The kit of the present invention prepares the knockout plasmid based on the CRISPR-CAS9 lentiviral vector by means of molecular cloning, that is, by constructing the guide RNA sequence of the target gene and recombining it into the linearized CRISPR-CAS9 lentiviral vector. The specific process as follows:

1. During the linearization process of the CRISPR-CAS9 lentiviral vector, the total volume should preferably be 50 μl, of which the CRISPR-CAS9 lentiviral vector is 2 μg, the BsmBI enzyme is 2 μl, the BsmBI enzyme buffer is 5 μl, and the rest is supplemented with sterilized deionized water to 50 μl. Then place it at 37°C for 1 hour, and then perform gel cutting and recovery. The specific enzyme cutting steps are as follows:

2 μg CRISPR-Cas9 lentiviral circular vector

2 μl BsmBI enzyme

5μl BsmBI enzyme buffer (10U/μl)

41 μl sterile deionized water

50μl total volume

After digestion, the carrier is subjected to electrophoresis on 1% agarose gel, stained with nucleic acid dye and observed under ultraviolet light. Cut out no more than 400 mg of 1% agarose gel, dissolve it at 55°C and recover the carrier. Carrier concentration determination.

The inventors adjusted the amount of the carrier and the amount of BsmBI enzyme, evaluated the enzyme cutting efficiency, and screened out the ratio with the highest enzyme cutting efficiency.

Screening of conditions during the preparation of linearized CRISPR-CAS9 lentiviral vector

Carrier amount (μg) BsmBI enzyme (μl) Total volume (μl) Digestion efficiency (%) 1 1 20 55 1 2 20 70 2 1 20 50 2 2 20 65 1 5 50 95 1 10 50 90 2 5 50 98 2 10 50 80

2. Construction of the guide RNA sequence of the target gene, gRNA target selection and oligonucleotide chaining (http://crispr-mit.edu/ website) sgRNA target selection and oligonucleotide chain synthesis: application design The standard is as follows: add CACC to the 5' end of the sense strand template; add AAAC to the 5' end of the antisense strand template.

Anneal the single strand of sgRNA oligonucleotides to form double strands: take equal amounts of sense strand and antisense strand and mix (final concentration is 10 μl). The program is as follows: 37°C, 30 minutes; 95°C, 5 minutes; 94°C, 12sec, -1°C/cycle, 70 cycles; finally 24°C, the specific reaction sample volume and concentration are as follows.

1 μl sense strand template (100mM)

1 μl antisense strand template (100mM)

1 μl T4 ligase buffer

7.5 μl sterile deionized water

10 μl total volume

3. For the recombination of the target gene and the linearized CRISPR-CAS9 lentiviral vector, dilute the sense and antisense strands obtained above to 0.5 μmoL/L. Add 10 ng of the linearized CRISPR-CAS9 vector obtained in (1), and add T4 ligase for ligation for 20 minutes. Transform highly transformable Stbl3 competent cells and pick single clones. The specific process is as follows:

1 μl linearized CRISPR-Cas9 vector 10ng

1 μl sense and antisense strand mix

1 μl T4 ligase buffer

1 μl T4 ligase (20U/μl)

Add sterile deionized water to a total volume of 10 μl.

The inventors changed the volume of T4 ligase and determined the amount of T4 ligase by ligation efficiency. The detection method of the ligation efficiency is to transform the bacteria after the ligation reaction, and compare the number of bacterial spots grown with the same content of the control plasmid.

Screening of T4 ligase and buffer combinations

T4 ligase volume (μl) Proportion of T4 ligase (%) Connection efficiency (%) 0.5 5 80 1 10 95 1.5 15 95 2 20 95

4. Lentiviral Vector Sequencing

The selected single clones were expanded and cultured in the Stbl3 bacterial culture medium, and ampicillin was added for resistance screening, the collected Stbl3 bacteria were lysed, and the recombinant plasmids were extracted for sequencing. The vectors that were successfully sequenced showed that the recombinant clones were constructed successfully.

Comparison experiment of Stbl3 competent and DH5 competent cells

The kit of the present invention is based on the linearized CRISPR-CAS9 lentiviral vector to construct a gene knockout kit with the following advantages:

(1) The linearized CRISPR-CAS9 lentiviral vector eliminates the inconsistency of different experimenters on the vector, reduces the interference of different reactions, saves the time of vector processing, and improves the experimental efficiency;

(2) The T4 ligase and buffer provided in the present invention are the optimal combination obtained through various tests, which avoids the difference in connection efficiency between different brands and improves the consistency of the experiment;

(3) The highly infectious Stbl3 competent cell constructed by the calcium chloride method of the present invention reduces the occurrence of recombination compared with the traditional DH5 competent cell.

4. With the present invention, the user only needs to synthesize the forward and reverse strand primers of the target gene to immediately construct the gene knockout vector, which has the effect of saving time, labor and cost.

Description of drawings

Figure 1 is an agarose gel electrophoresis image of the linearized CRISPR-CAS9 lentiviral vector, which shows the linearized vector and the fragments digested with BsmBI nuclease. Since there is a 2000bp insert sequence in the vector, the sequence is cut off after enzyme digestion, and the cut-off 2000bp sequence can prove to obtain a linearized CRISPR-CAS9 lentiviral vector, which is convenient for recovery.

Fig. 2 is the situation after the recombinant plasmid is plated after successfully transforming Stbl3.

Figure 3 shows the situation of transformation into cells, with green fluorescence showing successful transfection, which is convenient for subsequent experiments.

detailed description

The general cloning and recombination method described in the following example 1 is a gene knockout constructed by using a commonly used gene-established CRISPR-CAS9 lentiviral vector, and the gene knockout constructed by a CRISPR-CAS9 vector with a green fluorescent label on the carrier method.

Example 1 (CRISPR-CAS9 lentiviral vector gene knockout with puromycin resistance constructed by common cloning and recombination method, taking SNX10 gene as an example)

1. Mouse SNX10 gene sgRNA design:

(1) Design the positive and antisense strand primer sequences of the SNX10 gene:

sgRNA1F: AAACGCAACGCATTACTTTGGAGTC SEQ ID No.1

sgRNA1R: CACCGCACGTGGATCAGCGTCGCCA SEQ ID No. 2

sgRNA2F: CACCGCACGTGGATCAGCGTCGCCA SEQ ID No.3

sgRNA2R: AAACTGGCGACGCTGATCCACGTGC SEQ ID No.4

(2) The linearized CRISPR-CAS9 lentiviral vector with puromycin resistance described in the kit;

(3) T4 ligase and T4 ligase buffer;

(4) High transformation activity Stbl3 competent;

2. Recombination of CRISPR-CAS9 lentiviral cloning vector with puromycin resistance

(1) Anneal the sense strand and the antisense strand to form double strands: take equal amounts of the sense strand and the antisense strand and mix (final concentration is 10 μM). The program is as follows: 37°C for 30 minutes; 95°C for 5 minutes; 94°C for 12sec, -1°C/cycle, 70 cycles; the final temperature is 24°C. The specific reaction sample volume and concentration are as follows.

1 μl sense strand template (100mM)

1 μl antisense strand template (100mM)

1 μl T4 ligase buffer

Add sterile deionized water to a total volume of 10 μl.

After the reaction is complete, add 2 ml of sterilized deionized water to 10 μl.

(2) Add the sense strand and antisense strand mixture obtained above and the linearized CRISPR-CAS9 vector in the kit to the T4 ligase in the kit for ligation, and ligate for 20 minutes. The specific method is:

1 μl linearized puromycin-resistant CRISPR-Cas9 vector

1 μl sense and antisense strand mix

1 μl T4 ligase buffer

1 μl T4 ligase

Add sterile deionized water to a total volume of 10 μl.

(3) Thaw the Stbl3 competent cells with high transformation activity in this kit on ice, add the above 11 μl sense strand and antisense strand mixture into the competent cells, shake well, and place on ice for about 30 minutes. After heat-shocking in a water bath at 42°C for 90 seconds, add 1 ml of LB medium (sodium chloride 10g, peptone 10g; yeast extract 5g, add 1 liter of water to sterilize before use), and culture in a shaker at 37°C at 170 rpm After 1 hour, centrifuge at 5000 rpm for 1 minute. Spread the precipitate on an LB agarose (sodium chloride 10g, peptone 10g; yeast extract 5g, agar powder 3g, add 1 liter of water after sterilization) plate containing antibiotic activity, take it out after 16 hours of growth, and pick a single clone colony.

3. Sequencing of the CRISPR-CAS9 lentiviral cloning vector, adding the above-selected monoclonal colonies to LB medium for expansion culture, and adding ampicillin antibiotic (100g/ml) for resistance screening, 220 rpm at 37°C After culturing in a shaker for 12 hours, the plasmids were extracted and sequenced.

Sequencing results show that in the recombinant cloning plasmid, the above two positive-sense strand templates have been inserted into the vector:

The recombinant clone was constructed successfully.

Example 2 (CRISPR-CAS9 lentiviral vector gene knockout with puromycin resistance constructed by common cloning and recombination method, taking HSF1 gene as an example)

1. Human HSF1 gene sgRNA design:

(1) Design the positive and antisense strand primer sequences of the HSF1 gene:

sgRNA1F: TCGTGAGCGACCCGGACACCGTTTT SEQ ID No.5

sgRNA1R: GGTGTCCGGGTCGCTCACGACGGTG SEQ ID No. 6

sgRNA2F: CAGCTTCCACGTGTTCGACCGTTTT SEQ ID No. 7

sgRNA2R: GGTCGAACACGTGGAAGCTGCGGTG SEQ ID No.8

sgRNA3F: GGAGTCAATGAGGGCGGTCGGTTTT SEQ ID No. 9

sgRNA3R: CGACCGCCCTCATTGACTCCCGGTG SEQ ID No.10

(2) The linearized CRISPR-CAS9 lentiviral vector with puromycin resistance described in the kit;

(3) T4 ligase and T4 ligase buffer;

(4) High transformation activity Stbl3 competent;

2. Recombination of CRISPR-CAS9 lentiviral cloning vector with puromycin resistance

(1) Anneal the sense strand and the antisense strand to form double strands: take equal amounts of the sense strand and the antisense strand and mix (final concentration is 10 μM). The program is as follows: 37°C, 30 minutes; 95°C, 5 minutes; 94°C, 12sec, -1°C/cycle, 70 cycles; finally 24°C, the specific reaction sample volume and concentration are as follows.

1 μl sense strand template (100mM)

1 μl antisense strand template (100mM)

1 μl T4 ligase buffer

Add sterile deionized water to a total volume of 10 μl.

After the reaction is complete, add 2 ml of sterilized deionized water to 10 μl.

(2) Add the sense strand and antisense strand mixture obtained above and the linearized CRISPR-CAS9 vector in the kit to the T4 ligase in the kit for ligation, and ligate for 20 minutes. The specific method is:

1 μl linearized puromycin-resistant CRISPR-Cas9 vector

1 μl sense and antisense strand mix

1 μl T4 ligase buffer

1 μl T4 ligase

Add sterile deionized water to a total volume of 10 μl.

(3) Thaw the Stbl3 competent cells with high transformation activity in this kit on ice, add the above 11 μl sense strand and antisense strand mixture into the competent cells, shake well, and place on ice for about 30 minutes. After heat-shocking in a water bath at 42°C for 90 seconds, add 1 ml of LB medium (sodium chloride 10g, peptone 10g; yeast extract 5g, add 1 liter of water to sterilize before use), and culture in a shaker at 37°C at 170 rpm After 1 hour, centrifuge at 5000 rpm for 1 minute. Spread the precipitate on an LB agarose (sodium chloride 10g, peptone 10g; yeast extract 5g, agar powder 3g, add 1 liter of water after sterilization) plate containing antibiotic activity, take it out after 16 hours of growth, and pick a single clone colony.

3. Sequencing of the CRISPR-CAS9 lentiviral cloning vector, adding the above-selected monoclonal colonies to LB medium for expansion culture, and adding ampicillin antibiotic (100g/ml) for resistance screening, 220 rpm at 37°C After culturing in a shaker for 12 hours, the plasmids were extracted and sequenced.

Sequencing results show that in the recombinant cloning plasmid, the above two positive-sense strand templates have been inserted into the vector:

The recombinant clone was constructed successfully.

Example 3 (CRISPR-CAS9 lentiviral vector gene knockout with puromycin resistance constructed by common cloning and recombination method, taking PKA gene as an example)

1. Rat PKA gene sgRNA design:

(1) Design and obtain the positive and antisense strand primer sequences of the PKA gene:

sgRNA1F: AACGCCGCCGCCGCCAAGAAGTTTT SEQ ID No.11

sgRNA1R: TTCTTGGCGGCGGCGGCGTTCGGTG SEQ ID No.12

sgRNA2F: CCTCCCAATCCGCCGTAAGTGTTTT SEQ ID No. 13

sgRNA2R: ACTTACGGCGGATTGGGAGGCGGTG SEQ ID No.14

sgRNA3F: CGATCTGCGCCGCGTAGAAAGTTTT SEQ ID No. 15

sgRNA3R: TTTCTACGCGGCGCAGATCGCGGTG SEQ ID No. 16

(2) The linearized CRISPR-CAS9 lentiviral vector with puromycin resistance described in the kit;

(3) T4 ligase and T4 ligase buffer;

(4) High transformation activity Stbl3 competent;

2. Recombination of CRISPR-CAS9 lentiviral cloning vector with puromycin resistance

(1) Anneal the sense strand and the antisense strand to form double strands: take equal amounts of the sense strand and the antisense strand and mix (final concentration is 10 μM). The program is as follows: 37°C, 30 minutes; 95°C, 5 minutes; 94°C, 12sec, -1°C/cycle, 70 cycles; finally 24°C, the specific reaction sample volume and concentration are as follows.

1 μl sense strand template (100mM)

1 μl antisense strand template (100mM)

1 μl T4 ligase buffer

Add sterile deionized water to a total volume of 10 μl.

After the reaction is complete, add 2 ml of sterilized deionized water to 10 μl.

(2) Add the sense strand and antisense strand mixture obtained above and the linearized CRISPR-CAS9 vector in the kit to the T4 ligase in the kit for ligation, and ligate for 20 minutes. The specific method is:

1 μl linearized puromycin-resistant CRISPR-Cas9 vector

1 μl sense and antisense strand mix

1 μl T4 ligase buffer

1 μl T4 ligase

Add sterile deionized water to a total volume of 10 μl.

(3) Thaw the Stbl3 competent cells with high transformation activity in this kit on ice, add the above 11 μl sense strand and antisense strand mixture into the competent cells, shake well, and place on ice for about 30 minutes. After heat-shocking in a water bath at 42°C for 90 seconds, add 1 ml of LB medium (sodium chloride 10g, peptone 10g; yeast extract 5g, add 1 liter of water to sterilize before use), and culture in a shaker at 37°C at 170 rpm After 1 hour, centrifuge at 5000 rpm for 1 minute. Spread the precipitate on an LB agarose (sodium chloride 10g, peptone 10g; yeast extract 5g, agar powder 3g, add 1 liter of water after sterilization) plate containing antibiotic activity, take it out after 16 hours of growth, and pick a single clone colony.

3. Sequencing of the CRISPR-CAS9 lentiviral cloning vector, adding the above-selected monoclonal colonies to LB medium for expansion culture, and adding ampicillin antibiotic (100g/ml) for resistance screening, 220 rpm at 37°C After culturing in a shaker for 12 hours, the plasmids were extracted and sequenced.

Sequencing results show that in the recombinant cloning plasmid, the above two positive-sense strand templates have been inserted into the vector:

Recombinant clone 1: AAACGCAACGCATTACTTTGGAGTC SEQ ID No.17

Recombinant clone 2: CACCGCACGTGGATCAGCGTCGCCA SEQ ID No.18

The recombinant clone was constructed successfully.

SEQUENCE LISTING

<110> Southern Medical University

<120> A linearized CRISPR-CAS9 lentiviral vector gene knockout kit and its application

<160> 18

<170> PatentIn version 3.3

<210> 1

<211> 25

<212>DNA

<213> sgRNA1F

<400> 1

aaacgcaacg cattactttg gagtc 25

<210> 2

<211> 25

<212>DNA

<213> sgRNA1R

<400> 2

caccgcacgt ggatcagcgt cgcca 25

<210> 3

<211> 25

<212>DNA

<213> sgRNA2F

<400> 3

caccgcacgt ggatcagcgt cgcca 25

<210> 4

<211> 25

<212>DNA

<213> sgRNA2R

<400> 4

aaactggcga cgctgatcca cgtgc 25

<210> 5

<211> 25

<212>DNA

<213> sgRNA1F

<400> 5

tcgtgagcga cccggacacc gtttt 25

<210> 6

<211> 25

<212>DNA

<213> sgRNA1R

<400> 6

ggtgtccggg tcgctcacga cggtg 25

<210> 7

<211> 25

<212>DNA

<213> sgRNA2F

<400> 7

cagcttccac gtgttcgacc gtttt 25

<210> 8

<211> 25

<212>DNA

<213> sgRNA2R

<400> 8

ggtcgaacac gtggaagctg cggtg 25

<210> 9

<211> 25

<212>DNA

<213> sgRNA3F

<400> 9

ggagtcaatg agggcggtcg gtttt 25

<210> 10

<211> 25

<212>DNA

<213> sgRNA3R

<400> 10

cgaccgccct cattgactcc cggtg 25

<210> 11

<211> 25

<212>DNA

<213> sgRNA1F

<400> 11

aacgccgccg ccgccaagaa gtttt 25

<210> 12

<211> 25

<212>DNA

<213> sgRNA1R

<400> 12

ttcttggcgg cggcggcgtt cggtg 25

<210> 13

<211> 25

<212>DNA

<213> sgRNA2F

<400> 13

cctcccaatc cgccgtaagt gtttt 25

<210> 14

<211> 25

<212>DNA

<213> sgRNA2R

<400> 14

acttacggcg gattgggagg cggtg 25

<210> 15

<211> 25

<212>DNA

<213> sgRNA3F

<400> 15

cgatctgcgc cgcgtagaaa gtttt 25

<210> 16

<211> 25

<212>DNA

<213> sgRNA3R

<400> 16

tttctacgcg gcgcagatcg cggtg 25

<210> 17

<211> 25

<212>DNA

<213> recombinant clone 1

<400> 17

aaacgcaacg cattactttg gagtc 25

<210> 18

<211> 25

<212>DNA

<213> recombinant clone 2

<400> 18

caccgcacgt ggatcagcgt cgcca 25

Claims (7)

1. A gene recombination kit based on CRISPR-CAS9, which kit includes CRISPR-CAS9 carrier, T4 ligase and competent cells, characterized in that said CRISPR-CAS9 carrier is a linear CRISPR-CAS9 carrier, preferably Lentiviral, plasmid or adenoviral vectors. 2. The gene recombination kit according to claim 1, wherein the linear CRISPR-CAS9 vector is obtained by digesting the CRISPR-CAS9 lentiviral circular vector with BsmBI enzyme, and recovering by agarose gel; wherein the 2 μg of CRISPR-CAS9 lentiviral circular vector and 50 U of BsmBI enzyme in every 50 μl of the digested reaction solution. 3. The gene recombination kit according to claim 1, wherein said competent cells are Stbl3 competent cells, preferably Stbl3 competent cells treated with calcium chloride. 4. The genetic recombination kit according to claim 1, further comprising a T4 ligase buffer, and the dosage of the T4 ligase is 20 U T4 ligase per 10 ng of the linearized CRISPR-CAS9 vector. 5. The gene recombination kit according to claim 1, wherein also comprising the sense strand template and the antisense strand template of the sgRNA oligonucleotide of the target gene, CACC is added to the 5' end of the sense strand template; Add AAAC to the 5' end. 6. The genetic recombination kit according to claim 1, comprising 10ng linear CRISPR-CAS9 lentiviral vector, 1-2 μl 20U/μl T4 ligase, 1 μl T4 ligase buffer and Stbl3 competent cells. 7. A method for carrying out gene recombination with the gene recombination kit described in any one of claims 1-6, comprising the steps of: 1) annealing the sense strand template and the antisense strand template of the sgRNA oligonucleotide to form a double strand; 2) performing a ligation reaction with the sense strand and antisense strand mixture obtained in step 1) and the linearized CRISPR-CAS9 vector in the gene recombination kit with T4 ligase, to obtain the sgRNA oligonucleotide carrier connected to the target gene; 3) The carrier of the sgRNA oligonucleotide connected to the target gene is cultured as competent cells, and monoclonal colonies are obtained by culture medium.