CN114854791A - Novel CRISPR-Cas9 system vector and application thereof - Google Patents

Abstract

The embodiment of the invention relates to the technical field of biology, in particular to a novel CRISPR-Cas9 system vector and application thereof. The carrier provided by the embodiment of the invention sequentially comprises the following functional elements: an upstream LoxP sequence, a sgRNA sequence, a Cas9 protein, an inducible promoter, a Cre recombinase and a downstream LoxP sequence in the same direction; wherein the sgRNA sequence and Cas9 protein sequences are interchangeable; the sgRNA sequence targets the gene of interest. According to the vector provided by the invention, a CRISPR-Cas9 system and a Cre-Loxp system are combined, the gene knockout functions of the two systems are fully exerted, and an upstream LoxP sequence is arranged in front of the CRISPR-Cas9 system; sequentially arranging an inducible promoter, a Cre recombinase and a downstream homodromous LoxP sequence behind the CRISPR-Cas9 system; after the CRISPR-Cas9 system completes the gene knockout or gene knock-in function, the inducible promoter is induced to start expressing Cre recombinase, the Cre-Loxp system is started, the functional element between the upstream LoxP sequence and the downstream LoxP sequence is knocked out, and the Cre-Loxp system and the CRISPR-Cas9 system are completely prevented from excessively shearing cell genomes or causing immune reaction.

Description

A Novel CRISPR-Cas9 System Vector and Its Application

technical field

The invention relates to the field of biotechnology, in particular to a novel CRISPR-Cas9 system vector and its application.

Background technique

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology is a novel gene editing method that can be used for gene-directed knockout, and has attracted wide attention in the field of gene therapy. Cas9 endonuclease cuts double-stranded DNA under the guidance of guide ribonucleic acid (guideRNA, sgRNA), causing double-stranded breaks in the genome, and using the instability of cellular genome repair to generate non-specific recombination to generate repair errors (insertion or deletion), thereby Frameshift mutations may result in the loss of gene function and achieve the purpose of gene knockout.

At present, many researchers use the CRISPR-Cas9 system to knock out disease-causing genes or knock-in beneficial genes for disease prevention or treatment, but they have not considered the potential dangers of the CRISPR-Cas9 system to cells after gene knock-out or knock-in: First, As an exogenous protein, Cas9 protein may cause an immune response in the body; secondly, sgRNA as an exogenous nucleic acid in the cell may also cause an immune response; thirdly, the coexistence of Cas9 protein and sgRNA may also cause excessive splicing of the cell genome cut.

Although there are cases using RNA transfection technology to directly co-transfect sgRNA and Cas9 mRNA into cells to be transformed for transient expression, these technologies are mostly used in in vitro experiments and are not suitable for in vivo experiments or in vivo gene editing therapy.

Cre-loxP is a site-specific gene recombination technology. Cre protein (Cyclization Recombination Enzyme) is a recombinant enzyme consisting of 343 amino acids. The LoxP site (locus of X-overP1) is a 34bp sequence located in the P1 phage. It consists of two 13bp reverse palindromic sequences and an 8bp intermediate spacer sequence. The spacer sequence determines the direction of loxP. Cre recombinase can recognize LoxP sites and cause gene recombination between two LoxP sites. Cre recombinase can act on DNA substrates of various structures, such as linear, circular and even supercoiled DNA, without any cofactors, but Cre recombinase may also cause immune responses in vivo.

The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

SUMMARY OF THE INVENTION

Purpose of invention

In order to solve the above technical problems, the purpose of the present invention is to provide a novel CRISPR-Cas9 system vector and its application. The invention combines the CRISPR-Cas9 system with the Cre-Loxp system to give full play to the gene knockout functions of the two systems, and sets the upstream LoxP sequence before the CRISPR-Cas9 system; after the CRISPR-Cas9 system, an inducible promoter, Cre recombinase and the downstream LoxP sequence in the same direction; after the CRISPR-Cas9 system completes the gene knockout or gene knock-in function, the inducible promoter is induced to express Cre recombinase, the Cre-Loxp system is activated, and the upstream LoxP sequence Knockout of functional elements between the downstream homologous LoxP sequences completely avoids the Cre-Loxp system and the CRISPR-Cas9 system over-splicing the cell genome or causing immune responses.

solution

In order to achieve the purpose of the present invention, an embodiment of the present invention provides a carrier, which sequentially includes the following functional elements:

Upstream LoxP sequence, sgRNA sequence, Cas9 protein, inducible promoter, Cre recombinase, and downstream LoxP sequence in the same direction; sgRNA sequence and Cas9 protein sequence are interchangeable; sgRNA sequence targets the target gene. A spacer sequence from 0 to several hundred bp may be included between each functional element.

In a possible implementation manner of the above vector, the vector further includes a constitutive promoter before the sgRNA sequence and the Cas9 protein, respectively, and a transcription termination element after the Cas9 protein.

In a possible implementation manner of the above-mentioned vector, the upstream LoxP sequence includes the sequence shown in SEQ ID NO.1 or the complementary sequence of the sequence shown in SEQ ID NO.1, and the direct downstream LoxP sequence includes the sequence shown in SEQ ID NO.9. The sequence shown in SEQ ID NO.9 or the complementary sequence of the sequence shown in SEQ ID NO.9; or the upstream LoxP sequence includes the sequence shown in SEQ ID NO.24 or the complementary sequence of the sequence shown in SEQ ID NO.24, and the downstream LoxP sequence in the same direction includes SEQ ID NO.24. The sequence shown in NO.25 or the complementary sequence of the sequence shown in SEQ ID NO.25; or the upstream LoxP sequence includes the sequence shown in SEQ ID NO.28 or the complementary sequence of the sequence shown in SEQ ID NO.28, and the downstream LoxP sequence in the same direction Include the sequence shown in SEQ ID NO.29 or the complementary sequence of the sequence shown in SEQ ID NO.29.

In a possible implementation manner of the above vector, the constitutive promoter includes CMV, MSCV or U6 promoter; alternatively, the U6 promoter sequence is such as SEQ ID NO.6.

In a possible implementation of the above-mentioned carrier, the Cas9 protein includes saCas9, spCas9, AsCpf1, Cjcas9, NmCas9, St1Cas9 or TdCas9; alternatively, the Cas9 protein includes saCas9; further alternatively, the saCas9 protein sequence is such as SEQ ID NO. 3 shown.

In a possible implementation manner of the above-mentioned vector, the inducible promoter includes Mx1, TRE, tTA or ER promoter; alternatively, the inducible promoter includes Mx1 or TRE promoter; further optionally, the Mx1 promoter sequence As shown in SEQ ID NO.7; the TRE promoter sequence is shown in SEQ ID NO.23.

In a possible implementation of the above vector, the transcription termination element includes bGH polyA; alternatively, the bGH polyA sequence is shown in SEQ ID NO.5.

In a possible implementation mode of the above-mentioned carrier, Cre recombinase includes Escherichia virus P1Cre recombinase, Sphingomonas sp.ERG5 Cre recombinase, or Salmonella phage SJ46 Cre recombinase; alternatively Escherichia virus P1 Cre recombinase, which The coding sequence is shown in SEQ ID NO.8.

In a possible implementation manner of the above vector, the sgRNA targets the p53 gene; optionally, the sgRNA sequence includes the sequence shown in SEQ ID NO. 2 or the complementary sequence of the sequence shown in SEQ ID NO. 2.

In a possible implementation manner of the above vector, the sgRNA targets the RPGR gene; optionally, the sgRNA sequence includes the sequence shown in SEQ ID NO.18 or the complementary sequence of the sequence shown in SEQ ID NO.18.

In a possible implementation manner of the above-mentioned vector, the vector includes a plasmid.

In a possible implementation manner of the above-mentioned vector, the vector includes a viral vector; optionally, the viral vector includes a lentiviral vector, a retroviral vector, an adenoviral vector or an adeno-associated viral vector.

The embodiments of the present invention also provide cells comprising the vector; optionally, the cells include human cells, non-human mammalian cells, and stem cells.

The embodiments of the present invention also provide a composition, which includes the carrier and pharmaceutically acceptable excipients.

The embodiments of the present invention also provide applications of the above-mentioned vector, cells comprising the above-mentioned vector, and the above-mentioned composition in the preparation of a gene modification or gene expression regulation kit or medicine.

In a possible implementation manner of the above application, the modification is a knockout.

An embodiment of the present invention also provides a kit, the kit includes: the above-mentioned carrier, and/or the above-mentioned composition, and/or the above-mentioned cell.

beneficial effect

At present, although the Cre-Loxp system and the CRISPR-Cas9 system are used together, they only use the Cre-Loxp system to regulate the expression of the CRISPR-Cas9 system in space and time, and have no effect on gene knockout or knock-in. The CRISPR-Cas9 system was used for removal, and the Cre-Loxp system was also not effectively removed. Cre recombinase may cause an immune response in the body.

The vector provided by the present invention combines the CRISPR-Cas9 system with the Cre-Loxp system, fully exerts the gene knockout function of the two systems, and sets the upstream LoxP sequence before the CRISPR-Cas9 system; Induce the promoter, Cre recombinase and the downstream LoxP sequence in the same direction; after the CRISPR-Cas9 system completes the gene knockout or gene knockin function, induce the inducible promoter, start to express the Cre recombinase, and the Cre-Loxp system starts, The functional elements between the upstream LoxP sequence and the downstream LoxP sequence in the same direction are knocked out to completely avoid the Cre-Loxp system and the CRISPR-Cas9 system over-splicing the cell genome or causing an immune response.

Description of drawings

One or more embodiments are exemplified by the pictures in the corresponding drawings, and these exemplified descriptions do not constitute limitations on the embodiments. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

FIG. 1 is a schematic structural diagram of a functional element in Example 1 of the present invention.

Figure 2 is the electrophoresis diagram of the detection of p53 gene in 293T cells transfected with pCDH-MSCV-sa-sgRNA1-Cre plasmid and control 293T cells in Example 1 of the present invention, wherein: mock represents the control 293T cell group, and sg1 represents the transfection Group of 293T cells with pCDH-MSCV-sa-sgRNA1-Cre plasmid.

Fig. 3 is the electrophoretogram of detecting the knockout effect of cas9 after adding IFN-β and without adding IFN-β to 293T cells transfected with pCDH-MSCV-sa-sgRNA1-Cre plasmid in Example 1 of the present invention, wherein:- IFN represents a control group without IFN-β added, and +IFN represents a 293T cell group added with IFN-β.

Fig. 4 is the cDNA PCR result of eye retinal cells injected or not injected with lentivirus in the mouse in Example 2 of the present invention, mock represents the uninjected virus group, and the RPGR cDNA amplification product is 105bp; +virus group is the lentivirus injection group, and there is no corresponding Bands.

Figure 5 is the western result of the total protein of the retinal cells of the eyeballs injected or not injected with lentivirus in Example 2 of the present invention. Mock represents the non-virus injection group, and RPGR protein is visible; the +virus group is the lentivirus injection group, and there is no corresponding band.

Fig. 6 is the cDNA PCR result of the retinal cells of the eyeball after injecting or not injecting lentivirus and injecting or not injecting IFN-β in Example 2 of the present invention. Mouse B was injected with 100IU of IFN-β in the fundus retina to induce the expression of sgRNA2; Mice C were not injected with IFN-β, as a control; -virus means no lentivirus injected, +virus means lentivirus injected.

Figure 7 is the western result of the total protein of retinal cells in the eyeballs injected or not injected with lentivirus and injected with or without IFN-β in Example 2 of the present invention. Mouse B was injected with 100IU of IFN-β in the fundus retina to induce the expression of sgRNA2 ; Mice C were not injected with IFN-β, as a control; -virus means no lentivirus injected, +virus means lentivirus injected.

Figure 8 is the electrophoresis diagram of the detection of p53 gene in 293T cells transfected with pCDH-MSCV-sa-sgRNA1-Cre2 plasmid and control 293T cells in Example 3 of the present invention, wherein: mock represents the control 293T cell group, and sg1 represents the transfection Group of 293T cells with pCDH-MSCV-sa-sgRNA1-Cre2 plasmid.

Figure 9 shows that the 293T cells transfected with the pCDH-MSCV-sa-sgRNA1-Cre2 plasmid and the control 293T cells in Example 3 of the present invention were respectively added with a final concentration of 1 μg/ml or without Doxyclcline (Dox) to induce Cre2 recombinase. Genomic PCR detection electrophoresis of Cas9 in PCR products, in which: mock represents control 293T cell group, sg1 represents 293T cell group transfected with pCDH-MSCV-sa-sgRNA1-Cre2 plasmid, Dox+ represents induction by adding Doxyclcline, Dox- represents Doxyclcline was not added for induction.

Figure 10 shows that in Example 3 of the present invention, 293T cells transfected with pCDH-MSCV-sa-sgRNA1-Cre2 plasmid and control 293T cells were respectively added with a final concentration of 1 μg/ml or without Doxyclcline (Dox) to induce Cre recombinase. The results of western detection of cas protein expression, in which: mock indicates the control 293T cell group, sg1 indicates the 293T cell group transfected with pCDH-MSCV-sa-sgRNA1-Cre2 plasmid, Dox+ indicates induction by adding Dox, Dox- indicates induction without Dox .

Figure 11 is the electrophoresis diagram of the detection of p53 gene in 293T cells transfected with pCDH-MSCV-sa-sgRNA1-Cre3 plasmid and control 293T cells in Example 4 of the present invention, wherein: mock represents the control 293T cell group, and sg1 represents the transfection Group of 293T cells with pCDH-MSCV-sa-sgRNA1-Cre3 plasmid.

Figure 12 is the electrophoresis image of the detection of Cas9 products by PCR after the 293T cells transfected with the pCDH-MSCV-sa-sgRNA1-Cre3 plasmid in Example 4 of the present invention after induction with or without IFN-β, respectively, wherein: +IFN means IFN-β was added for induction, -IFN means no IFN-β was added for induction.

Figure 13 shows the results of western detection of cas protein expression in 293T cells transfected with pCDH-MSCV-sa-sgRNA1-Cre3 plasmid and control 293T cells with or without IFN-β to induce Cre recombinase in Example 4 of the present invention , where: mock represents the control 293T cell group, sg1 represents the 293T cell group transfected with pCDH-MSCV-sa-sgRNA1-Cre3 plasmid, IFN+ represents induction by adding IFN-β, and IFN- represents induction without IFN-β.

Figure 14 is the electrophoresis diagram of the detection of p53 gene in 293T cells transfected with pCDH-MSCV-sa-sgRNA1-Cre4 plasmid and control 293T cells in Example 5 of the present invention, wherein: mock represents the control 293T cell group, and sg1 represents the transfection Group of 293T cells with pCDH-MSCV-sa-sgRNA1-Cre4 plasmid.

Figure 15 is the electrophoresis image of the detection of Cas9 product by PCR detection of 293T cells transfected with pCDH-MSCV-sa-sgRNA1-Cre4 plasmid in Example 5 of the present invention after induction with or without IFN-β, wherein: +IFN means IFN-β was added for induction, -IFN means no IFN-β was added for induction.

Figure 16 shows the results of western detection of cas protein expression in 293T cells transfected with pCDH-MSCV-sa-sgRNA1-Cre4 plasmid and control 293T cells with or without IFN-β to induce Cre recombinase in Example 5 of the present invention , where: mock represents the control 293T cell group, sg1 represents the 293T cell group transfected with pCDH-MSCV-sa-sgRNA1-Cre4 plasmid, IFN+ represents induction by adding IFN-β, and IFN- represents induction without IFN-β.

Figure 17 is a schematic structural diagram of the functional element of the FG12-sa-sgRNA1-Cre2 vector in Example 6 of the present invention.

Figure 18 is the detection electrophoresis diagram of p53 gene in 293T cells transfected with FG12-sa-sgRNA1-Cre2 plasmid and control 293T cells in Example 6 of the present invention, wherein: mock represents the control 293T cell group, and sg1 represents the transfected FG12 - 293T cell group of sa-sgRNA1-Cre2 plasmid.

Figure 19 is the electrophoresis image of the detection of Cas9 product by PCR after the 293T cells transfected with the FG12-sa-sgRNA1-Cre2 plasmid were induced with or without Doxyclcline (Dox) in Example 6 of the present invention, wherein: +Dox means adding Doxyclcline induction, -Dox means no Doxyclcline addition.

Detailed ways

In order to make the purposes, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of them. example. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. Unless expressly stated otherwise, throughout the specification and claims, the term "comprising" or its conjugations such as "comprising" or "comprising" and the like will be understood to include the stated elements or components, and Other elements or other components are not excluded.

In addition, in order to better illustrate the present invention, numerous specific details are given in the following detailed description. It will be understood by those skilled in the art that the present invention may be practiced without certain specific details. In some embodiments, materials, elements, methods, means, etc. that are well known to those skilled in the art are not described in detail, so as to highlight the gist of the present invention.

The molecular cloning part in the examples of the present invention, such as the experimental steps of enzyme digestion, ligation, PCR, gel electrophoresis, gel recovery, transformation, transfection, etc., can all refer to the "Molecular Cloning Experiment Guide (Fourth Edition)" (Science Publishing) Society, J. Sambrook, M.R. Green) related chapters.

Example 1

1. A carrier, referred to as pCDH-MSCV-sa-sgRNA1-Cre in the implementation of the present invention, is: a multi-cloning site in a lentivirus shuttle plasmid (model is pCDH-MSCV-MCS-EF1-copGFP) A vector inserted with the following functional elements from 5'-3' including:

The upstream LoxP sequence, saCas9 protein, bGH polyA sequence, U6 promoter, sgRNA sequence, Mx1 promoter, Cre recombinase, and direct downstream LoxP sequence; its schematic diagram is shown in Figure 1.

Wherein, the upstream LoxP sequence is: 5'-ATAAC TTCGT ATAGC ATACA TTATA CGAAG TTAT-3' (SEQ ID NO.1);

In this embodiment, the sgRNA is sgRNA1, which targets the p53 gene, and the sequence is: AGCAA AGTTT TATTGTAAAA T (SEQ ID NO. 2);

The saCas9 protein sequence is shown in SEQ ID NO.3; its cDNA sequence is shown in SEQ ID NO.4;

The bGH polyA sequence is shown in SEQ ID NO.5;

The U6 promoter sequence is shown in SEQ ID NO.6;

The Mx1 promoter sequence is shown in SEQ ID NO.7;

The Cre recombinase cDNA sequence is shown in SEQ ID NO.8;

The direct downstream LoxP sequence is: 5'-ATAAC TTCGT ATAGC ATACA TTATA CGAAG TTAT-3' (SEQ ID NO. 9).

The construction method of the lentiviral shuttle plasmid pCDH-MSCV-sa-sgRNA1-Cre includes the following steps (the PX601 vector used in the experiment is a commercialized vector, purchased from XYbscience):

1) The PX601 vector was digested with the restriction endonuclease BsaI to obtain a linearized plasmid.

2) Two oligonucleotide fragments (oligos) of sgRNA1 were synthesized artificially, annealed, and inserted into the linearized PX601 vector to obtain a PX601-sgRNA1 vector.

The sequences of the two oligonucleotide fragments of the synthesized sgRNA1 are as follows:

Oligo1-5': CACCG AGCAA AGTTT TATTG TAAAA T (SEQ ID NO. 10);

Oligo1-3': AAACA TTTTA CAATA AAACT TTGCT C (SEQ ID NO. 11).

3) The "CMV-saCas9-bGH polyA-U6-sgRNA1" element is PCR-amplified from the PX601-sgRNA1 vector, wherein the upstream and downstream primers used for amplification respectively contain upstream and downstream LoxP sequences, and the primer sequences are as follows:

LoxP-Cas9-F:

TCTTGAAAGGAGTGGGAATTCTCGAGGCGTTATAACTTCGTATAGCATACATTATACGAAGTTATGACATTGATTATTGACTAGT (SEQ ID NO. 12);

LoxP-Cas9-R:

GCTAAGATCTACAGCTGCCTCGGCCGCAAAAATCTCGCCA (SEQ ID NO. 13); the amplified product is represented by "LoxP-CMV-saCas9-bGH polyA-U6-sgRNA1-LoxP".

4) The pCDH-MSCV-MCS-EF1-copGFP vector was digested with restriction enzymes AgeI and KpnI to obtain a linearized plasmid.

5) The aforementioned PCR product "LoxP-CMV-saCas9-bGH polyA-U6-sgRNA1-LoxP" is connected to the linearized pCDH-MSCV-MCS-EF1-copGFP plasmid by the method of homologous recombination, and the obtained plasmid is named as pCDH -MSCV-LoxP-CMV-saCas9-U6-sgRNA1.

6) The pCDH-MSCV-loxP-CMV-saCas9-U6-sgRNA1 plasmid was digested with restriction endonuclease NotI to obtain a linearized plasmid.

7) Using the method of homologous recombination, the artificially synthesized "MX1 (SEQ ID NO.7)-Cre (SEQ ID NO.8)-downstream LoxP (SEQ ID NO.9)" element was connected into the linearized pCDH-MSCV -LoxP-CMV-saCas9-U6-sgRNA1 plasmid, the resulting vector was named pCDH-MSCV-sa-sgRNA1-Cre vector.

2. Transfection of 293T cells

The constructed lentiviral shuttle plasmid pCDH-MSCV-sa-sgRNA1-Cre was transfected into 293T cells.

Specific steps are as follows:

1) 24h before transfection, trypsinize 293T cells in logarithmic growth phase, adjust the cell density to 1.2×10 ⁷ cells/20ml with medium containing 10% serum, and re-seek them in a 15cm cell culture dish at 37°C, Cultured in a 5% CO ₂ incubator. After culturing for 24 hours, the cells can be used for transfection when the cell density reaches 70% to 80%. The cell state is critical for viral packaging, so good cell state and low number of passages need to be guaranteed. The cell culture medium was changed to serum-free medium 2 h before transfection.

2) Add 2 μg of pCDH-MSCV-sa-sgRNA1-Cre vector to a sterilized centrifuge tube, and mix with 100 μl of Opti-MEM.

4) Gently shake the Lipofectamine 2000 reagent, take 6 μl of Lipofectamine 2000 reagent and mix it with 100 μl of Opti-MEM in another tube, and incubate at room temperature for 5 minutes.

5) Mix the diluted DNA with the diluted Lipofectamine 2000, invert and mix gently, do not shake, and must mix within 5 minutes.

6) After mixing, incubate for 20 minutes at room temperature to form transfection complexes of DNA and Lipofectamine 2000 dilution.

7) Transfer the mixture of DNA and Lipofectamine 2000 to the culture medium of 293T cells, mix well, and culture in a 37°C, 5% CO ₂ cell incubator.

8) Divide the untransfected 293T and the transfected 293T into three 35mm culture dishes 1:3 after culturing for 8 hours, change 2ml of cell culture medium with 10% serum, and continue in a 37°C, 5% _CO2 incubator. Incubate for 48 hours.

3. Detection of knockout effect of p53 gene

The PCR method was used to detect the copy number of the p53 gene in the genome, and the detection steps were as follows:

1) Take a 35mm culture dish of transfected 293T cells and untransfected 293T cells as a control to extract the genome;

2) Use detection primers to detect the copy number of p53 in the genome;

The detection primers used are: p53-F1: GGCCC ACCTC TTACC GATT T (SEQ ID NO. 14);

p53-R1: CAGGA GCCAT TGTCT TTGAG G (SEQ ID NO. 15).

The detection results are shown in Figure 2. As can be seen from Figure 2, the gene product of wild-type p53 (717bp) can be seen in the control group (represented by "mock" in Figure 2); while the transfected pCDH-MSCV-sa-sgRNA1-Cre The group (indicated by "sg1" in Figure 2) has no specific band at the corresponding position, indicating that sgRNA1 played a gene knockout function, and the p53 gene was successfully knocked out.

4. Detection of knockout effect of cas9

After detection of p53 knockout, Cre recombinase expression was induced using IFN-β:

1) Add 500IU of recombinant IFN-β to 293T cells transfected with pCDH-MSCV-sa-sgRNA1-Cre in a 35mm dish; 293T cells transfected with pCDH-MSCV-sa-sgRNA1-Cre in another 35mm dish Cells were used as a control group without IFN-β.

2) After 72h, extract the genomes of two plates of 293T cells;

3) Use detection primers to detect the copy number of cas9:

The specific PCR primers used were:

cas9-F2: AAGAG CAGAT CAGCC GGAAC (SEQ ID NO. 16);

cas9-R2: AAGCT TCTCT TCACG ACGGG (SEQ ID NO. 17);

The detection results are shown in Figure 3. It can be seen from Figure 3 that the PCR product of cas9 (935bp) can be seen in the control group (without IFN-β group, indicated by "-IFN" in the figure); while the IFN-β group ( There is no specific band in the corresponding position of "+IFN" in the figure, indicating that cas9 was successfully knocked out after induction by IFN-β. That is, the Cre-Loxp system is activated, and the functional elements between the upstream LoxP sequence and the downstream LoxP sequence in the same direction are successfully knocked out, which can completely avoid the Cre-Loxp system and the CRISPR-Cas9 system.

Example 2

1. A vector, referred to as pCDH-MSCV-sa-sgRNA2-Cre in the implementation of the present invention, the structure and construction method of the lentiviral shuttle vector are the same as those in Example 1, the difference is only in the sgRNA sequence.

The sgRNA used in this example is sgRNA2, which targets the RPGR gene, and its sequence is: CCTTT CCCAT GTACTGTTTC (SEQ ID NO. 18).

Its construction steps are the same as those in Example 1, except that two oligonucleotide fragments (oligos) of sgRNA2 are artificially synthesized, annealed, and inserted into the BsaI linearized PX601 carrier to obtain a PX601-sgRNA2 carrier; the remaining steps are the same as in the example. 1 is the same, the obtained vector is named pCDH-MSCV-sa-sgRNA2-Cre;

The sequences of the two oligonucleotide fragments of the artificially synthesized sgRNA2 are as follows:

Oligo2F: CACCG CCTTT CCCAT GTACT GTTTC (SEQ ID NO. 19);

Oligo2R: AAAC GAAAC AGTAC ATGGG AAAGGC (SEQ ID NO. 20).

2. Packaging lentivirus

The constructed lentivirus shuttle plasmid pCDH-MSCV-sa-sgRNA2-Cre was used to package the lentivirus; the obtained lentivirus was injected into the retina of the mouse. Specific steps are as follows:

(1) Cell transfection

1) 24h before transfection, trypsinize 293T cells in logarithmic growth phase, adjust the cell density to 1.2×10 ⁷ cells/20ml with medium containing 10% serum, and re-seek them in a 15cm cell culture dish at 37°C, Cultured in a 5% CO ₂ incubator. After culturing for 24 hours, the cells can be used for transfection when the cell density reaches 70% to 80%. The cell state is critical for viral packaging, so good cell state and low number of passages need to be guaranteed.

2) The cell culture medium was changed to serum-free medium 2 h before transfection.

3) Add each prepared DNA solution (20 μg of pCDH-MSCV-sa-sgRNA2-Cre carrier, 15 μg of pHelper 1.0 carrier, 10 μg of pHelper 2.0 carrier) into a sterilized centrifuge tube, and mix with the corresponding volume of Opti-MEM, Adjust the total volume to 2.5 ml and incubate for 5 minutes at room temperature.

4) Gently shake the Lipofectamine 2000 reagent, take 100 μl of Lipofectamine 2000 reagent and mix it with 2.4 ml of Opti-MEM in another tube, and incubate at room temperature for 5 minutes.

5) Mix the diluted DNA with the diluted Lipofectamine 2000, invert and mix gently, do not shake, and must mix within 5 minutes.

6) After mixing, incubate for 20 minutes at room temperature to form transfection complexes of DNA and Lipofectamine 2000 dilution.

7) Transfer the mixture of DNA and Lipofectamine 2000 to the culture medium of 293T cells, mix well, and culture in a 37°C, 5% CO ₂ cell incubator.

8) After culturing for 8 hours, the medium containing the transfection mixture was poured out, 20 ml of PBS was added to each flask of cells, and the culture flask was gently shaken from side to side to wash the remaining transfection mixture, and then poured out.

9) Add 25ml of cell culture medium containing 10% serum to each flask of cells, and continue to culture for 48 hours in a 37°C, 5% CO ₂ incubator.

(2) Harvest and concentration of virus

1) Collect the supernatant of 293T cells 48 hours after transfection (the transfection can be counted from 0 hours).

2) Centrifuge at 4000g for 10min at 4°C to remove cell debris.

3) Filter the supernatant with a 0.45 μm filter into a 40 ml ultracentrifuge tube.

4) Add the virus crude extract sample to the filter cup (up to 19ml) and close the lid. Insert the filter cup into the filtrate collection tube.

5) After the combination is completed, balance it and place it on the rotor.

6) Centrifuge at 4000 x g to the desired virus concentration volume. It usually takes 10-15 minutes.

7) After the centrifugation, take out the centrifuge device and separate the filter cup from the filtrate collection cup below.

8) Invert the filter cup over the sample collection cup.

9) The centrifugal force does not exceed 1000g, and the time is 2 minutes. Excessive rotational speeds can result in sample loss. Remove the centrifuge cup from the sample collection cup. What is in the sample collection cup is the virus concentrate.

10) Remove the virus concentrate, store it in a virus tube after packaging, and store it at -80°C for a long time. One of them was used for viral biological titer determination, and the viral titer was quantitatively characterized by ELISA p24.

3. Mouse fundus retinal injection

Three mice were selected as the experimental group, numbered as A, B, and C, respectively. One eyeball of each mouse was used as a control without virus injection, and the other side was injected with virus.

The subretinal injection method is:

1) After preparing the corresponding experimental items, the mice were dilated with 1% atropine, and then anesthetized by intraperitoneal injection of 80 mg/kg ketamine + 8 mg/kg xylazine.

2) After anesthesia, the pupils were dilated again, and then the mice were placed in front of the animal experimental platform of the ophthalmic surgery microscope, and 0.5% proparacaine was locally anesthetized on the mouse eyeballs.

3) Add fluorescein sodium stock solution to 400ng p24 virus at a concentration of 100:1, mix with a pipette, be careful not to have air bubbles. 4) Use an insulin needle to pre-poke a small hole in the pars plana of the mouse eyeball, and use the needle of a microsyringe to pass through the small hole and enter the mouse eyeball vitreous cavity. At this time, drop an appropriate amount of 2% on the mouse eyeball. Hydroxymethylcellulose can clearly see the fundus of the mouse under the microscope, and then continue to insert the needle into the retina of the contralateral periphery, avoiding the lens, and slowly push the virus with sodium fluorescein, the injection volume of each eye is 1 μl , using sodium fluorescein as an indicator to judge whether the injection was injected into the subretinal space.

5) Observe whether the mice are abnormal after operation, wash the surface of the eyeball with normal saline, and place it in a cage to wake up from anesthesia. Neomycin eye ointment can be given to prevent infection according to the situation of the mice.

4. Detection of RPGR gene expression

After 2 weeks, mouse A was taken to detect the expression of RPGR protein in retinal cells; the specific steps were as follows:

1) Take mouse eyeballs, separate retinal cells, and divide them into two parts, one for extracting total protein and one for extracting RNA;

2) RNA was reverse transcribed to obtain cDNA, and RT-PCR was used to detect the expression of RPGR gene;

The primers used for the detection of the RPGR gene were:

RPGR-F3: TGGCG ACTTT TCTGC CGTAT (SEQ ID NO. 21);

RPGR-R3: AATCT GGTTC CTCTG GCTGC (SEQ ID NO. 22);

The detection results are shown in Figure 4. It can be seen from Figure 4 that the cDNA amplification product of RPGR (258bp) can be seen in the uninjected virus group (represented by "mock" in Figure 4), while the injected virus group (represented by "mock" in Figure 4) +virus" indicates) no corresponding band.

3) Western Blot was used for the total protein of the extracted mouse retinal cells to detect the expression level of RPGR protein; α-actin (actin) was used as an internal reference;

The detection results are shown in Figure 5. As can be seen from Figure 5, the non-virus group (indicated by "mock" in Figure 5) showed normal expression of RPGR protein, while the expression of RPGR protein in the injected virus group (indicated by "+virus" in Figure 5) was significantly attenuated. Figures 4 and 5 illustrate that sgRNA2 played a gene knockout function in mouse eyeballs, and the RPGR gene was successfully knocked out.

5. Detection of cas9 and RPGR gene expression

After detection of RPGR gene knockout, mouse B was injected with 100 IU IFN-β into the fundus and retina to induce Cre recombinase expression; mouse C was not injected with IFN-β as a control.

After 1 week, the sequence between the two LoxP sequences in retinal cells was detected by PCR, and the detection steps were as follows:

1) Take the eyeballs of mouse B and mouse C, separate retinal cells, and divide them into two parts, one for extracting total protein and one for extracting RNA;

2) RNA was reverse transcribed to obtain cDNA, and RT-PCR was used to detect the expression of cas9 and RPGR genes;

The primers used to detect cas9 are the same as those in Example 1, which are: cas9-F2 (SEQ ID NO. 16) and cas9-R2 (SEQ ID NO. 17)

The primers used for detection of RPGR are the same as above: RPGR-F3 (SEQ ID NO. 21) and RPGR-R3 (SEQ ID NO. 22).

The detection results are shown in Figure 6. It can be seen from Figure 6 that the cDNA amplification product of RPGR (258bp) can be seen in the uninjected virus group (represented by "-virus" in Figure 6) with or without IFN-β induction. , and there is no PCR product of cas9 (935bp).

In the virus injection group (represented by "+virus" in Figure 6), with or without IFN-β induction, there was no cDNA amplification product of RPGR (258bp); without IFN-β induction, cas9 (935bp) was seen. After IFN induction was added, there was no PCR product of cas9, that is, after IFN induction was added, cas9 was successfully removed.

3) Western Blot was used for the total protein of the extracted mouse retinal cells to detect the expression levels of cas9 and RPGR proteins, and the endogenous antibody α-RPGR of RPGR and the tag antibody anti-HA of cas9 were used for color development; α-actin ( actin) as an internal reference;

The test results are shown in Figure 7. It can be seen from Figure 7 that the non-injected virus group (represented by "-virus" in Figure 7) with or without IFN-β induction showed that the RPGR protein was expressed normally, and there was no cas9 protein. Express.

In the virus injection group (represented by "+virus" in Figure 7), with or without IFN-β induction, the expression of RPGR was significantly reduced; without IFN-β induction, the expression of cas9 protein was seen; with IFN-β added After induction, the expression of cas9 protein was not seen, that is, after induction by IFN, cas9 was successfully knocked out.

Figures 6 and 7 illustrate that in the mouse eyeball, after IFN induction, the Cre-Loxp system is activated, and the functional elements between the upstream LoxP sequence and the downstream LoxP sequence in the same direction are successfully knocked out, which can completely avoid the Cre-Loxp system and The CRISPR-Cas9 system over-cuts the cellular genome or elicits an immune response.

Example 3

1. A vector, called pCDH-MSCV-sa-sgRNA1-Cre2 in the implementation of the present invention, the structure of the lentiviral shuttle vector is the same as that of Example 1, the difference is only in the inducible promoter.

In this example, the inducible promoter used is TRE, the sequence of which is shown in SEQ ID NO.23.

The construction steps refer to Example 1, the difference is that the synthetic "TRE (SEQ ID NO. 23)-Cre (SEQ ID NO. 8)-downstream LoxP (SEQ ID NO. 9)" element MSCV-LoxP-CMV-saCas9-U6-sgRNA1 plasmid, the obtained vector was named pCDH-MSCV-sa-sgRNA1-Cre2 vector.

The pCDH-MSCV-sa-sgRNA1-Cre2 vector was transfected into 293T cells to detect the knockout effect of p53. The experimental steps were the same as those in Example 1, and the results are shown in Figure 8 .

In Figure 8, genomes were extracted from control 293T cells that were not transfected with pCDH-MSCV-sa-sgRNA1-Cre2 vector and 293T cells transfected with pCDH-MSCV-sa-sgRNA1-Cre2 vector, using the detection primer p53- F1 and p53-R1 detect the copy number of p53 in the genome. mock represents the control 293T cell group, sg1 represents the 293T cell group transfected with the pCDH-MSCV-sa-sgRNA1-Cre2 plasmid, the control group shows the wild-type p53 gene product of 717bp, transfected with pCDH-MSCV-sa-sgRNA1-Cre2 There is no specific band in the corresponding position of 293T cells of the vector. This indicates that the p53 gene is knocked out.

After detecting that the p53 gene was knocked out, two plates were prepared for the control 293T cells not transfected with the pCDH-MSCV-sa-sgRNA1-Cre2 vector and the 293T cells transfected with the pCDH-MSCV-sa-sgRNA1-Cre2 vector, respectively. The final concentration of 1μg/ml or no Doxyclcline (Dox) was used to induce Cre recombinase expression, and the genome was extracted after 72h; the cas9 detection primers cas9-F2 and cas9-R2 were used to detect the copy number of Cas9, the results are shown in Figure 9; at the same time, 72h After that, the total cell protein was extracted, and the Western Blot method was used to detect the expression level of cas9 protein, and the cas9 tag antibody anti-HA was used for color development; α-actin (actin) was used as an internal reference, and the results were shown in Figure 10.

As can be seen from Figure 9, the control 293T (represented by mock in the figure) without the transfection of the pCDH-MSCV-sa-sgRNA1-Cre2 vector has no PCR product of Cas9 regardless of whether Doxyclcline (Dox) is added; When the 293T cells of MSCV-sa-sgRNA1-Cre2 vector (represented by sg1 in the figure) were not induced by Dox, they had the PCR product of Cas9, but there was no specific band of Cas9 in the corresponding position of the Dox group, indicating that after induction by Dox , the Cre-LoxP system played a gene knockout function and successfully knocked out the cas9 gene.

As can be seen from Figure 10, the control 293T (represented by mock in the figure) without the transfection of the pCDH-MSCV-sa-sgRNA1-Cre2 vector has no cas9 protein expression whether or not Doxyclcline (Dox) is added; transfected with pCDH-MSCV -sa-sgRNA1-Cre2 vector 293T cells (represented by sg1 in the figure) had cas9 protein expression when Dox was not added for induction, but no cas9 protein was detected in the Dox group, indicating that after Dox induction, Cre-LoxP system The gene knockout function was exerted, and the cas9 gene was successfully knocked out, but no cas9 protein was expressed.

Example 4

1. A vector, called pCDH-MSCV-sa-sgRNA1-Cre3 in the implementation of the present invention, the structure of the lentiviral shuttle vector is the same as that in Example 1, the only difference is the LoxP sequence.

In this example, the upstream LoxP-2 sequence used is: ATAAC TTCGT ATAAT GTATG CTATA CGAAGTTAT (SEQ ID NO.24);

The downstream LoxP-2 sequence is: ATAAC TTCGT ATAAT GTATG CTATA CGAAG TTAT (SEQ ID NO. 25).

The construction of the lentiviral shuttle plasmid pCDH-MSCV-sa-sgRNA1-Cre3 was completed on the basis of the pCDH-MSCV-MCS-EF1-copGFP vector and the pCDH-MSCV-sa-sgRNA1-Cre vector in Example 1. The build method includes the following steps:

1) The pCDH-MSCV-MCS-EF1-copGFP vector was digested with restriction enzymes AgeI and KpnI to obtain a linearized plasmid.

2) The "CMV-saCas9-bGH polyA-U6-sgRNA1-Mx1-Cre" element is obtained by PCR amplification from the pCDH-MSCV-sa-sgRNA1-Cre carrier, wherein the upstream and downstream primers used for the amplification include upstream and downstream respectively LoxP-2 sequence, the upstream and downstream primers used are:

LoxP-2-Cas9-F:

TCTTGAAAGGAGTGGGAATTCTCGAGGCGTTATAACTTCGTATAATGTATGCTATACGAAGTTATGACATTGATTATTGACTAGT (SEQ ID NO. 26);

Cre-loxP-2-R:

GGAGTGAATTAGCCCTTCCAATAACTTCGTATAGCATACATTATACGAAGTTATCTAATCGCCATCTTCCAGCA (SEQ ID NO. 27); the amplified product is represented by "LoxP2-CMV-saCas9-bGH polyA-U6-sgRNA1-Mx1-Cre-LoxP2".

3) The aforementioned PCR product "LoxP2-CMV-saCas9-bGH polyA-U6-sgRNA1-Mx1-Cre-LoxP2" was connected to the linearized pCDH-MSCV-MCS-EF1-copGFP plasmid by the method of homologous recombination, and the obtained The plasmid was named pCDH-MSCV-sa-sgRNA1-Cre3.

The pCDH-MSCV-sa-sgRNA1-Cre3 vector was transfected into 293T cells to detect the knockout effect of p53. The experimental steps were the same as those in Example 1, and the results are shown in Figure 11 .

In Figure 11, the genomes were extracted from the control 293T cells not transfected with the pCDH-MSCV-sa-sgRNA1-Cre3 vector and the 293T cells transfected with the pCDH-MSCV-sa-sgRNA1-Cre3 vector, using the detection primer p53- F1 and p53-R1 detect the copy number of p53 in the genome. mock represents the control 293T cell group, sg1 represents the 293T cell group transfected with the pCDH-MSCV-sa-sgRNA1-Cre3 plasmid, the control group shows the wild-type p53 gene product of 717bp, transfected with pCDH-MSCV-sa-sgRNA1-Cre3 There is no specific band in the corresponding position of 293T cells of the vector. This indicates that the p53 gene is knocked out.

After detecting that the p53 gene was knocked out, two plates were prepared for the control 293T cells that were not transfected with the pCDH-MSCV-sa-sgRNA1-Cre3 vector and the 293T cells that were transfected with the pCDH-MSCV-sa-sgRNA1-Cre3 vector. The expression of Cre recombinase was induced by 500IU or without recombinant IFN-β, and the genome was extracted after 72h; the copy number of Cas9 was detected by using cas9 detection primers cas9-F2 and cas9-R2, and the results are shown in Figure 12; Protein, the Western Blot method was used to detect the expression level of cas9 protein, and the cas9 tag antibody anti-HA was used for color development; α-actin (actin) was used as an internal reference, and the results were shown in Figure 13.

It can be seen from Figure 12 that when the 293T cells transfected with the pCDH-MSCV-sa-sgRNA1-Cre3 vector were not induced with IFN-β, they had the PCR product of Cas9, and the corresponding position of the IFN-β group was not specific for Cas9. Sexual bands, indicating that after induction by IFN-β, the Cre-LoxP system played a gene knockout function and successfully knocked out the cas9 gene.

As can be seen from Figure 13, the control 293T (represented by mock in the figure) that was not transfected with the pCDH-MSCV-sa-sgRNA1-Cre3 vector did not induce IFN-β and had no cas9 protein expression; transfected with pCDH-MSCV-sa -sgRNA1-Cre3 vector 293T cells (indicated by sg1 in the figure) expressed cas9 protein when they were not induced with IFN-β, but no cas9 protein was detected in the IFN-β group, indicating that after induction with IFN-β, The Cre-LoxP system played a gene knockout function and successfully knocked out the cas9 gene without cas9 protein expression.

Example 5

1. A vector, called pCDH-MSCV-sa-sgRNA1-Cre4 in the implementation of the present invention, the lentiviral shuttle vector is the same as that in Example 1, the only difference is the LoxP sequence.

In this example, the upstream LoxP-3 sequence used is: ATAACTTCGTATA GGGTAGGCTATACGAAGTTAT (SEQ ID NO.28);

The downstream LoxP-3 sequence is: ATAACTTCGTATA GGGTAGGC TATACGAAGTTAT (SEQ ID NO. 29).

The construction of the lentiviral shuttle plasmid pCDH-MSCV-sa-sgRNA1-Cre4 was completed on the basis of the pCDH-MSCV-MCS-EF1-copGFP vector and the pCDH-MSCV-sa-sgRNA1-Cre vector in Example 1. The build method includes the following steps:

1) The pCDH-MSCV-MCS-EF1-copGFP vector was digested with restriction enzymes AgeI and KpnI to obtain a linearized plasmid.

2) The "CMV-saCas9-bGH polyA-U6-sgRNA1-Mx1-Cre" element is obtained by PCR amplification from the pCDH-MSCV-sa-sgRNA1-Cre carrier, wherein the upstream and downstream primers used for the amplification include upstream and downstream respectively LoxP-3 sequence, the upstream and downstream primers used are:

LoxP-3-Cas9-F:

GTGGGAATTCTCGAGGCGTTATAACTTCGTATAGGGTAGGCTATACGAAGTTATGACATTGATTATTGACTAGT (SEQ ID NO. 30);

Cre-loxP-3-R:

GGAGTGAATTAGCCCTTCCAATAACTTCGTATAGCCTACCCTATACGAAGTTATCTAATCGCCATCTTCCAGCA (SEQ ID NO. 31); the amplified product is represented by "LoxP3-CMV-saCas9-bGH polyA-U6-sgRNA1-Mx1-Cre-LoxP3". .

3) The aforementioned PCR product "LoxP3-CMV-saCas9-bGH polyA-U6-sgRNA1-Mx1-Cre-LoxP3" was connected to the linearized pCDH-MSCV-MCS-EF1-copGFP plasmid by the method of homologous recombination, and the obtained The plasmid was named pCDH-MSCV-sa-sgRNA1-Cre4.

The pCDH-MSCV-sa-sgRNA1-Cre4 vector was transfected into 293T cells to detect the knockout effect of p53. The experimental steps were the same as in Example 1, and the results are shown in Figure 14 .

In Figure 14, genomes were extracted from control 293T cells that were not transfected with pCDH-MSCV-sa-sgRNA1-Cre4 vector and 293T cells transfected with pCDH-MSCV-sa-sgRNA1-Cre4 vector, using the detection primer p53- F1 and p53-R1 detect the copy number of p53 in the genome. mock represents the control 293T cell group, sg1 represents the 293T cell group transfected with the pCDH-MSCV-sa-sgRNA1-Cre4 plasmid, the control group shows the wild-type p53 gene product of 717bp, transfected with pCDH-MSCV-sa-sgRNA1-Cre4 There is no specific band in the corresponding position of 293T cells of the vector. This indicates that the p53 gene is knocked out.

After it was detected that the p53 gene was knocked out, two plates were prepared for the control 293T cells that were not transfected with the pCDH-MSCV-sa-sgRNA1-Cre4 vector and the 293T cells that were transfected with the pCDH-MSCV-sa-sgRNA1-Cre4 vector. 500IU or no recombinant IFN-β was used to induce Cre recombinase expression, and the genome was extracted after 72h; the copy number of Cas9 was detected by using cas9 detection primers cas9-F2 and cas9-R2, and the results are shown in Figure 15; at the same time, the total number of cells was extracted after 72h. Protein, the Western Blot method was used to detect the expression level of cas9 protein, and the cas9 tag antibody anti-HA was used for color development; α-actin (actin) was used as an internal reference, and the results were shown in Figure 16.

It can be seen from Figure 15 that when the 293T cells transfected with the pCDH-MSCV-sa-sgRNA1-Cre4 vector were not induced with IFN-β, they had the PCR product of Cas9, and the corresponding position of the IFN-β group was not specific for Cas9. Sexual bands, indicating that after induction by IFN-β, the Cre-LoxP system played a gene knockout function and successfully knocked out the cas9 gene.

As can be seen from Figure 16, the control 293T (represented by mock in the figure) that was not transfected with the pCDH-MSCV-sa-sgRNA1-Cre3 vector did not induce IFN-β and had no cas9 protein expression; transfected with pCDH-MSCV-sa -sgRNA1-Cre3 vector 293T cells (indicated by sg1 in the figure) expressed cas9 protein when they were not induced with IFN-β, but no cas9 protein was detected in the IFN-β group, indicating that after induction with IFN-β, The Cre-LoxP system played a gene knockout function and successfully knocked out the cas9 gene without cas9 protein expression.

Example 6

1. A carrier, referred to as FG12-sa-sgRNA1-Cre2 in the implementation of the present invention, is: a multi-cloning site with the following functional elements inserted into the lentivirus shuttle plasmid (model is FG12, Addgene#14884); The vector, the functional element from 5'-3' includes:

The upstream LoxP sequence, saCas9 protein, bGH polyA sequence, U6 promoter, sgRNA sequence, TRE promoter, Cre recombinase, and direct downstream LoxP sequence; its schematic diagram is shown in FIG. 17 .

Wherein, the sequence of each element: upstream LoxP sequence, saCas9 protein, bGH polyA sequence, U6 promoter, sgRNA sequence, Cre recombinase, and the same direction downstream LoxP sequence as in Example 1;

The TRE promoter sequence is shown in SEQ ID NO.23.

The construction of the lentiviral shuttle plasmid FG12-sa-sgRNA1-Cre2 was completed on the basis of FG12 vector (Addgene#14884) and pCDH-MSCV-sa-sgRNA1-Cre2 vector. The build method includes the following steps:

1) The FG12 vector was digested with restriction endonucleases XhoI and BsrGI to obtain a linearized plasmid.

2) The "CMV-saCas9-bGH polyA-U6-sgRNA1-TRE-Cre" element is obtained by PCR amplification from the pCDH-MSCV-sa-sgRNA1-Cre2 vector, wherein the upstream and downstream primers used in the amplification respectively include upstream and downstream LoxP sequences, primer sequences are as follows:

FG12-loxP-2-Cas9-F:

AGTTAACATCTCGAGGCGTTATAACTTCGTATAGCATACATTATACGAAGTTATGACATTGATTATTGACTAGT (SEQ ID NO. 32);

FG12-Cre-loxP-2-R:

GGACTAGAGTCGCGGCCGCTATAACTTCGTATAATGTATGCTATACGAAGTTATCTAATCGCCATCTTCCAGCA (SEQ ID NO. 33); the amplified product is represented by "LoxP-CMV-saCas9-bGH polyA-U6-sgRNA1-TRE-Cre-LoxP".

3) Connect the aforementioned PCR product "LoxP-CMV-saCas9-bGH polyA-U6-sgRNA1-TRE-Cre-LoxP" into the linearized FG12 vector by the method of homologous recombination, and the obtained vector is named FG12-sa- sgRNA1-Cre2 vector.

The FG12-sa-sgRNA1-Cre2 vector was transfected into 293T cells to detect the knockout effect of p53. The experimental steps were the same as those in Example 1, and the results are shown in Figure 18.

In Figure 18, genomes were extracted from control 293T cells that were not transfected with FG12-sa-sgRNA1-Cre2 vector and 293T cells transfected with FG12-sa-sgRNA1-Cre2 vector, using the detection primers p53-F1 and p53- R1 detects the copy number of p53 in the genome. mock represents the control 293T cell group, sg1 represents the 293T cell group transfected with the FG12-sa-sgRNA1-Cre2 plasmid, the wild-type p53 gene product of 717bp in the control group, and 293T cells transfected with the FG12-sa-sgRNA1-Cre2 vector There is no specific band at the corresponding position. This indicates that the p53 gene is knocked out.

After detecting that the p53 gene was knocked out, two plates were prepared for the 293T cells transfected with the FG12-sa-sgRNA1-Cre2 vector, respectively adding a final concentration of 1 μg/ml or not adding Doxyclcline (Dox) to induce Cre recombinase expression, after 72h Extract the genome; use the cas9 detection primers cas9-F2 and cas9-R2 to detect the copy number of Cas9, the results are shown in Figure 19.

It can be seen from Figure 19 that when the 293T cells transfected with the FG12-sa-sgRNA1-Cre2 vector were not induced by Dox, they had the PCR product of Cas9. After Dox induction, the Cre-LoxP system played a gene knockout function and successfully knocked out the cas9 gene.

In the embodiment of the present invention, the cas9 protein can be saCas9, spCas9, AsCpf1, Cjcas9, NmCas9, St1Cas9, TdCas9. The Cre recombinase can be Escherichia virus P1 Cre recombinase, Sphingonas sp.ERG5 Cre recombinase, or Salmonella phage SJ46 Cre recombinase. The Cre-LoxP system can also be replaced by the Flp-FRT system. Those skilled in the art can select different enzymes according to actual needs. Different enzymes can be used in the present invention to carry out the present invention.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand: it can still be Modifications are made to the technical solutions described in the foregoing embodiments, or some technical features thereof are equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Beijing Zhongyin Technology Co., Ltd.

<120> A Novel CRISPR-Cas9 System Vector and Its Application

<130> 190300F

<160> 33

<170> PatentIn version 3.5

<210> 1

<211> 34

<212> DNA

<213> Artifical sequence

<400> 1

ataacttcgt atagcataca ttatacgaag ttat 34

<210> 2

<211> 21

<212> DNA

<213> Artifical sequence

<400> 2

agcaaagttt tattgtaaaa t 21

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<211> 1085

<212> PRT

<213> Staphylococcus aureus

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Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala

1 5 10 15

Ala Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser Val

20 25 30

Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly

35 40 45

Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg

50 55 60

Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile

65 70 75 80

Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His

85 90 95

Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu

100 105 110

Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu

115 120 125

Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr

130 135 140

Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala

145 150 155 160

Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys

165 170 175

Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr

180 185 190

Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln

195 200 205

Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg

210 215 220

Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys

225 230 235 240

Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe

245 250 255

Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr

260 265 270

Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn

275 280 285

Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe

290 295 300

Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu

305 310 315 320

Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys

325 330 335

Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr

340 345 350

Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala

355 360 365

Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu

370 375 380

Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser

385 390 395 400

Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile

405 410 415

Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala

420 425 430

Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln

435 440 445

Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro

450 455 460

Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile

465 470 475 480

Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg

485 490 495

Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys

500 505 510

Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr

515 520 525

Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp

530 535 540

Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu

545 550 555 560

Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro

565 570 575

Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys

580 585 590

Gln Glu Glu Asn Ser Lys Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu

595 600 605

Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile

610 615 620

Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu

625 630 635 640

Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp

645 650 655

Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu

660 665 670

Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys

675 680 685

Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp

690 695 700

Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp

705 710 715 720

Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys

725 730 735

Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys

740 745 750

Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu

755 760 765

Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp

770 775 780

Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile

785 790 795 800

Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu

805 810 815

Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu

820 825 830

Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His

835 840 845

Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly

850 855 860

Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr

865 870 875 880

Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile

885 890 895

Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp

900 905 910

Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr

915 920 925

Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val

930 935 940

Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser

945 950 955 960

Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala

965 970 975

Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly

980 985 990

Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile

995 1000 1005

Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn

1010 1015 1020

Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser

1025 1030 1035

Lys Thr Gln Ser Ile Lys Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn

1040 1045 1050

Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys

1055 1060 1065

Gly Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys

1070 1075 1080

Lys Lys

1085

<210> 4

<211> 3255

<212> DNA

<213> Artifical sequence

<400> 4

atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc caagcggaac 60

tacatcctgg gcctggacat cggcatcacc agcgtgggct acggcatcat cgactacgag 120

acacgggacg tgatcgatgc cggcgtgcgg ctgttcaaag aggccaacgt ggaaaacaac 180

gagggcaggc ggagcaagag aggcgccaga aggctgaagc ggcggaggcg gcatagaatc 240

cagagagtga agaagctgct gttcgactac aacctgctga ccgaccacag cgagctgagc 300

ggcatcaacc cctacgaggc cagagtgaag ggcctgagcc agaagctgag cgaggaagag 360

ttctctgccg ccctgctgca cctggccaag agaagaggcg tgcacaacgt gaacgaggtg 420

gaagaggaca ccggcaacga gctgtccacc aaagagcaga tcagccggaa cagcaaggcc 480

ctggaagaga aatacgtggc cgaactgcag ctggaacggc tgaagaaaga cggcgaagtg 540

cggggcagca tcaacagatt caagaccagc gactacgtga aagaagccaa acagctgctg 600

aaggtgcaga aggcctacca ccagctggac cagagcttca tcgacaccta catcgacctg 660

ctggaaaccc ggcggaccta ctatgaggga cctggcgagg gcagcccctt cggctggaag 720

gacatcaaag aatggtacga gatgctgatg ggccactgca cctacttccc cgaggaactg 780

cggagcgtga agtacgccta caacgccgac ctgtacaacg ccctgaacga cctgaacaat 840

ctcgtgatca ccagggacga gaacgagaag ctggaatatt acgagaagtt ccagatcatc 900

gagaacgtgt tcaagcagaa gaagaagccc accctgaagc agatcgccaa agaaatcctc 960

gtgaacgaag aggatattaa gggctacaga gtgaccagca ccggcaagcc cgagttcacc 1020

aacctgaagg tgtaccacga catcaaggac attaccgccc ggaaagagat tattgagaac 1080

gccgagctgc tggatcagat tgccaagatc ctgaccatct accagagcag cgaggacatc 1140

caggaagaac tgaccaatct gaactccgag ctgacccagg aagagatcga gcagatctct 1200

aatctgaagg gctataccgg cacccacaac ctgagcctga aggccatcaa cctgatcctg 1260

gacgagctgt ggcacaccaa cgacaaccag atcgctatct tcaaccggct gaagctggtg 1320

cccaagaagg tggacctgtc ccagcagaaa gagatcccca ccaccctggt ggacgacttc 1380

atcctgagcc ccgtcgtgaa gagaagcttc atccagagca tcaaagtgat caacgccatc 1440

atcaagaagt acggcctgcc caacgacatc attatcgagc tggcccgcga gaagaactcc 1500

aaggacgccc agaaaatgat caacgagatg cagaagcgga accggcagac caacgagcgg 1560

atcgaggaaa tcatccggac caccggcaaa gagaacgcca agtacctgat cgagaagatc 1620

aagctgcacg acatgcagga aggcaagtgc ctgtacagcc tggaagccat ccctctggaa 1680

gatctgctga acaacccctt caactatgag gtggaccaca tcatccccag aagcgtgtcc 1740

ttcgacaaca gcttcaacaa caaggtgctc gtgaagcagg aagaaaacag caagaagggc 1800

aaccggaccc cattccagta cctgagcagc agcgacagca agatcagcta cgaaaccttc 1860

aagaagcaca tcctgaatct ggccaagggc aagggcagaa tcagcaagac caagaaagag 1920

tatctgctgg aagaacggga catcaacagg ttctccgtgc agaaagactt catcaaccgg 1980

aacctggtgg ataccagata cgccaccaga ggcctgatga acctgctgcg gagctacttc 2040

agagtgaaca acctggacgt gaaagtgaag tccatcaatg gcggcttcac cagctttctg 2100

cggcggaagt ggaagtttaa gaaagagcgg aacaaggggt acaagcacca cgccgaggac 2160

gccctgatca ttgccaacgc cgatttcatc ttcaaagagt ggaagaaact ggacaaggcc 2220

aaaaaagtga tggaaaacca gatgttcgag gaaaagcagg ccgagagcat gcccgagatc 2280

gaaaccgagc aggagtacaa agagatcttc atcacccccc accagatcaa gcacattaag 2340

gacttcaagg actacaagta cagccaccgg gtggacaaga agcctaatag agagctgatt 2400

aacgacaccc tgtactccac ccggaaggac gacaagggca acaccctgat cgtgaacaat 2460

ctgaacggcc tgtacgacaa ggacaatgac aagctgaaaa agctgatcaa caagagcccc 2520

gaaaagctgc tgatgtacca ccacgacccc cagacctacc agaaactgaa gctgattatg 2580

gaacagtacg gcgacgagaa gaatcccctg tacaagtact acgaggaaac cgggaactac 2640

ctgaccaagt actccaaaaa ggacaacggc cccgtgatca agaagattaa gtattacggc 2700

aacaaactga acgcccatct ggacatcacc gacgactacc ccaacagcag aaacaaggtc 2760

gtgaagctgt ccctgaagcc ctacagattc gacgtgtacc tggacaatgg cgtgtacaag 2820

ttcgtgaccg tgaagaatct ggatgtgatc aaaaaagaaa actactacga agtgaatagc 2880

aagtgctatg aggaagctaa gaagctgaag aagatcagca accaggccga gtttatcgcc 2940

tccttctaca acaacgatct gatcaagatc aacggcgagc tgtatagagt gatcggcgtg 3000

aacaacgacc tgctgaaccg gatcgaagtg aacatgatcg acatcaccta ccgcgagtac 3060

ctggaaaaca tgaacgacaa gaggcccccc aggatcatta agacaatcgc ctccaagacc 3120

cagagcatta agaagtacag cacagacatt ctgggcaacc tgtatgaagt gaaatctaag 3180

aagcaccctc agatcatcaa aaagggcaaa aggccggcgg ccacgaaaaa ggccggccag 3240

gcaaaaaaga aaaag 3255

<210> 5

<211> 208

<212> DNA

<213> Artifical sequence

<400> 5

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180

gggaagagaa tagcaggcat gctgggga 208

<210> 6

<211> 241

<212> DNA

<213> Artifical sequence

<400> 6

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

c 241

<210> 7

<211> 1704

<212> DNA

<213> Artifical sequence

<400> 7

cctggatggg taagcagcca tgcaagggtt ccttgataat tgctggagaa gcctgtgttc 60

tcccattaga ctccagcaga gggccccata gaacatgcca gacttcaggc cagggagctg 120

ctctgtgtgg cctcctcccc ttcttttggg tgccaccaca ttgaaattgg ttccatccaa 180

gttttcccga tgtggctgaa gaatgtcacc agccatatca gtagacaaaa gaaattgcag 240

ccatcacgcc agcagccact gcattttttg acctgtgact gtacgtcctt caggggttca 300

ggatgaggaa aagcaggatg ctggccctag acagccaagg tgcacatcaa aggaataatt 360

tcaaggagcc cagactcttg catcttccca tgcatagaat aagtgctgaa ttcatgaact 420

tgggatgtct ggttttttctt taagtaacaa taactttgtt aatattttca ctacctggat 480

tttgttgcaa aaactcctat atgttctggc tcctctctga cctgtttgct gccgtcccta 540

agagcaatct gagaggctgt cttccagggt aagtcctcag caagtccacc aagtaaaata 600

taattctcaa ttttttttagg tgtgtagttt tttttttttta aagtcaacat tgtagcccag 660

cacttggaaa aggggcagtt ctgggtcttt gtgtaggggg caggtgaccc tctataacca 720

caggagtctt tgtgggggagg atacttgttg tccaggcttc ttagctgagc caaggaggat 780

ggaggtgggc tttccaaaat gaaattttac tcctcatccc caaagccctc cgggagggct 840

catctttggg tctatttgac cttggtcccc cactttctac tcatttccat cctgaagaaa 900

tcaaatgagc taaacttcag ggagggcttg ggacttccct ggtggtccag ggaacctttt 960

gcttccgatt cagggggcac aggtccgatc cctggtcaag aaaccaagat ctcacacgac 1020

atgcagagtg gtcaagagat ttaaaggtgg gtggaggggg cggtgcttga agaggataag 1080

gaaaattgag aaaggagcct gatgtaggtc tgggtgagga gggggctaag ggggtagctc 1140

cctagcatgc ttgagaatcc ctacgggcgc tggaatgttc ccagtgaacc tacagcagaa 1200

gtttgatacc caattatcaa tgcatctgtt caaacaacca aaggttaagg ttagccaggt 1260

tccaagctac cttcggcttt ggatgactcg gggctgcttg agcagaggtt ctcaaactgc 1320

cagaaacttc agaagggccg gataaagctc gggtagctgg gtcctactcc cgagtttctg 1380

gggcagcagg tctgggtgcg ggccgagaat ttgcatttcc cgcaagctcc tagggatgcc 1440

gttggtgcgg ggcgcaccta gagtgcttct gggaggatac agctgagggt gctgggcgca 1500

gcgacctcgg gaggcgccgg tgcgcaagtg cgctacccgt tcgatttggg tttcggtttc 1560

ctttccgatt cagcagccct gaaaactcta cgagtttcgt ttcccagagg ctgggtggga 1620

gatgacggac ggggaggcgg gggcagcgag ctgggggcgg cgctagcgct gcataaagcc 1680

gaggagggcc agcgccggga gccc 1704

<210> 8

<211> 1032

<212> DNA

<213> Artifical sequence

<400> 8

atgtccaatt tactgaccgt acaccaaaat ttgcctgcat taccggtcga tgcaacgagt 60

gatgaggttc gcaagaacct gatggacatg ttcagggatc gccaggcgtt ttctgagcat 120

acctggaaaa tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac 180

cggaaatggt ttcccgcaga acctgaagat gttcgcgatt atcttctata tcttcaggcg 240

cgcggtctgg cagtaaaaac tatccagcaa catttgggcc agctaaacat gcttcatcgt 300

cggtccgggc tgccacgacc aagtgacagc aatgctgttt cactggttat gcggcggatc 360

cgaaaagaaa acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact 420

gatttcgacc aggttcgttc actcatggaa aatagcgatc gctgccagga tatacgtaat 480

ctggcatttc tggggattgc ttataacacc ctgttacgta tagccgaaat tgccaggatc 540

agggttaaag atatctcacg tactgacggt gggagaatgt taatccatat tggcagaacg 600

aaaacgctgg ttagcaccgc aggtgtagag aaggcactta gcctgggggt aactaaactg 660

gtcgagcgat ggatttccgt ctctggtgta gctgatgatc cgaataacta cctgttttgc 720

cgggtcagaa aaaatggtgt tgccgcgcca tctgccacca gccagctatc aactcgcgcc 780

ctggaaggga tttttgaagc aactcatcga ttgatttacg gcgctaagga tgactctggt 840

cagagatacc tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg agatatggcc 900

cgcgctggag tttcaatacc ggagatcatg caagctggtg gctggaccaa tgtaaatatt 960

gtcatgaact atatccgtaa cctggatagt gaaacagggg caatggtgcg cctgctggaa 1020

gatggcgatt ag 1032

<210> 9

<211> 34

<212> DNA

<213> Artifical sequence

<400> 9

ataacttcgt atagcataca ttatacgaag ttat 34

<210> 10

<211> 26

<212> DNA

<213> Artifical sequence

<400> 10

caccgagcaa agtttttattg taaaat 26

<210> 11

<211> 26

<212> DNA

<213> Artifical sequence

<400> 11

aaacatttta caataaaact ttgctc 26

<210> 12

<211> 85

<212> DNA

<213> Artifical sequence

<400> 12

tcttgaaagg agtgggaatt ctcgaggcgt tataacttcg tatagcatac attatacgaa 60

gttatgacat tgattattga ctagt 85

<210> 13

<211> 40

<212> DNA

<213> Artifical sequence

<400> 13

gctaagatct acagctgcct cggccgcaaa aatctcgcca 40

<210> 14

<211> 20

<212> DNA

<213> Artifical sequence

<400> 14

ggcccacctc ttaccgattt 20

<210> 15

<211> 21

<212> DNA

<213> Artifical sequence

<400> 15

caggagccat tgtctttgag g 21

<210> 16

<211> 20

<212> DNA

<213> Artifical sequence

<400> 16

aagagcagat cagccggaac 20

<210> 17

<211> 20

<212> DNA

<213> Artifical sequence

<400> 17

aagcttctct tcacgacggg 20

<210> 18

<211> 20

<212> DNA

<213> Artifical sequence

<400> 18

ccttttcccat gtactgtttc 20

<210> 19

<211> 25

<212> DNA

<213> Artifical sequence

<400> 19

caccgccttt cccatgtact gtttc 25

<210> 20

<211> 25

<212> DNA

<213> Artifical sequence

<400> 20

aaacgaaaca gtacatggga aaggc 25

<210> 21

<211> 20

<212> DNA

<213> Artifical sequence

<400> 21

tggcgacttt tctgccgtat 20

<210> 22

<211> 20

<212> DNA

<213> Artifical sequence

<400> 22

aatctggttc ctctggctgc 20

<210> 23

<211> 247

<212> DNA

<213> Artifical sequence

<400> 23

tccctatcag tgatagagaa cgatgtcgag tttactccct atcagtgata gagaacgtat 60

gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga 120

tagagaacgt atgtcgagtt tatccctatc agtgatagag aacgtatgtc gagtttactc 180

cctatcagtg atagagaacg tatgtcgagg taggcgtgta cggtgggagg cctatataag 240

cagagct 247

<210> 24

<211> 34

<212> DNA

<213> Artifical sequence

<400> 24

ataacttcgt ataatgtatg ctatacgaag ttat 34

<210> 25

<211> 34

<212> DNA

<213> Artifical sequence

<400> 25

ataacttcgt ataatgtatg ctatacgaag ttat 34

<210> 26

<211> 85

<212> DNA

<213> Artifical sequence

<400> 26

tcttgaaagg agtgggaatt ctcgaggcgt tataacttcg tataatgtat gctatacgaa 60

gttatgacat tgattattga ctagt 85

<210> 27

<211> 74

<212> DNA

<213> Artifical sequence

<400> 27

ggagtgaatt agcccttcca ataacttcgt atagcataca ttatacgaag ttatctaatc 60

gccatcttcc agca 74

<210> 28

<211> 34

<212> DNA

<213> Artifical sequence

<400> 28

ataacttcgt atagggtagg ctatacgaag ttat 34

<210> 29

<211> 34

<212> DNA

<213> Artifical sequence

<400> 29

ataacttcgt atagggtagg ctatacgaag ttat 34

<210> 30

<211> 74

<212> DNA

<213> Artifical sequence

<400> 30

gtgggaattc tcgaggcgtt ataacttcgt atagggtagg ctatacgaag ttatgacatt 60

gattattgac tagt 74

<210> 31

<211> 74

<212> DNA

<213> Artifical sequence

<400> 31

ggagtgaatt agcccttcca ataacttcgt atagcctacc ctatacgaag ttatctaatc 60

gccatcttcc agca 74

<210> 32

<211> 74

<212> DNA

<213> Artifical sequence

<400> 32

agttaacatc tcgaggcgtt ataacttcgt atagcataca ttatacgaag ttatgacatt 60

gattattgac tagt 74

<210> 33

<211> 74

<212> DNA

<213> Artifical sequence

<400> 33

ggactagagt cgcggccgct ataacttcgt ataatgtatg ctatacgaag ttatctaatc 60

gccatcttcc agca 74

Claims (10)

1. A carrier comprising the following functional elements thereon in order:

an upstream LoxP sequence, a sgRNA sequence, a Cas9 protein, an inducible promoter, a Cre recombinase and a downstream LoxP sequence in the same direction; wherein the sgRNA sequence and Cas9 protein sequences are interchangeable; the sgRNA sequence targets the gene of interest.

2. The carrier of claim 1, wherein: on the carrier: the sgRNA sequence and the Cas9 protein each also include a constitutive promoter before and a transcription termination element after the Cas9 protein.

3. The carrier of claim 1 or 2, wherein: the upstream LoxP sequence comprises a sequence shown by SEQ ID NO.1 or a complementary sequence thereof, and the downstream LoxP sequence in the same direction comprises a sequence shown by SEQ ID NO.9 or a complementary sequence thereof; or the upstream LoxP sequence comprises a sequence shown in SEQ ID NO.24 or a complementary sequence thereof, and the downstream LoxP sequence comprises a sequence shown in SEQ ID NO.25 or a complementary sequence thereof; or the upstream LoxP sequence comprises a sequence shown by SEQ ID NO.28 or a complementary sequence thereof, and the downstream LoxP sequence comprises a sequence shown by SEQ ID NO.29 or a complementary sequence thereof.

4. The carrier of claim 1 or 2, wherein: inducible promoters include the Mx1, TRE, tTA or ER promoter; alternatively, the inducible promoter comprises the Mx1 or TRE promoter; further alternatively, the Mx1 promoter sequence is shown in SEQ ID NO. 7; the TRE promoter sequence is shown in SEQ ID NO. 23.

5. The carrier of claim 1 or 2, wherein: the sgRNA targets the p53 gene, and further optionally, the sgRNA sequence comprises a sequence shown in SEQ ID No.2 or a complementary sequence of the sequence shown in SEQ ID No. 2;

and/or the sgRNA targets the RPGR gene, further optionally the sgRNA sequence comprises the sequence shown in SEQ ID No.18 or a complement of the sequence shown in SEQ ID No. 18.

6. The carrier of claim 1 or 2, characterized in that: cas9 proteins include saCas9, spCas9, ascipf 1, Cjcas9, NmCas9, St1Cas9 or TdCas 9;

and/or, the Cre recombinase comprises Escherichia virus P1Cre recombinase, Sphingomonas sp.ERG5 Cre recombinase, or Salmonella phage SJ46 Cre recombinase; alternatively, the Cre recombinase comprises Escherichia virus P1Cre recombinase, the coding sequence of which is shown in SEQ ID NO. 8.

7. The carrier of claim 2, wherein: constitutive promoters include CMV, MSCV or U6 promoters;

and/or, the transcription termination element comprises bGH polyA; optionally, the bGH polyA sequence is as shown in SEQ ID No. 5;

and/or, the vector comprises a plasmid; optionally, the vector comprises a viral vector; further optionally, the viral vector comprises a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.

8. A cell comprising the vector of any one of claims 1-7, wherein: the cell comprises human cell, non-human mammal cell and stem cell.

9. A composition comprising the vector of any one of claims 1-7, and a pharmaceutically acceptable excipient.

10. Use of the vector of any one of claims 1-7, the cell of claim 8, the composition of claim 9 for the preparation of a kit for modifying or regulating gene expression or a medicament.

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