CN110004181B

CN110004181B - Additional CRISPR/Cas9 expression vector and construction method and application thereof

Info

Publication number: CN110004181B
Application number: CN201910212739.1A
Authority: CN
Inventors: 牟彦双; 李佳男; 刘忠华; 姜超前; 王玥; 矫彦文
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2019-03-20
Filing date: 2019-03-20
Publication date: 2022-10-11
Anticipated expiration: 2039-03-20
Also published as: CN110004181A

Abstract

An additional CRISPR/Cas9 expression vector and a construction method and application thereof belong to the field of cell engineering. In order to solve the problems that the existing CRISPR/Cas expression system is easy to integrate into a host cell genome and destroy the sequence in the genome, the invention provides an episomal CRISPR/Cas9 expression vector, which is a CRISPR/Cas9 expression vector containing an S/MAR sequence, and is a CRISPR/Cas9 expression vector containing an S/MAR sequence, wherein the nucleotide of the S/MAR sequence is shown as SEQ ID NO:1 is shown in the specification; the S/MAR sequence is located between the coding region sequence capable of coding for a protein and the bGH poly (A) sequence in this vector. The additional CRISPR/Cas9 expression vector provided by the invention can be used for dissociating and expressing Cas9 protein and single-stranded guide RNA outside a chromosome, and can be used for gene knockout research.

Description

Additional CRISPR/Cas9 expression vector and construction method and application thereof

Technical Field

The invention belongs to the technical field of cell engineering, and particularly relates to an additional CRISPR/Cas9 expression vector, and a construction method and application thereof.

Background

The CRISPR/Cas9 system is used as a novel powerful gene editing and genetic screening tool, and has wide application prospect in the aspects of biomedical fields such as establishment of disease models, gene therapy and the like through gene editing. The CRISPR/Cas9 system mainly comprises two members, a Cas9 protein and a single-stranded guide RNA (sgRNA) in the process of performing a gene editing function. The Cas9 protein and the single-stranded guide RNA are mainly obtained by transcription expression of an expression vector in tissue cells, and the commonly used expression vector is a retrovirus expression vector or a plasmid expression vector. However, the CRISPR/Cas9 system still faces some technical difficulties in practical application, and one of the problems is that a vector expressing the CRISPR/Cas9 system is easily integrated into a host cell genome after entering a host cell, so that the sequence in the genome is damaged, and a potential safety hazard exists.

Disclosure of Invention

In order to solve the problems that the existing CRISPR/Cas expression system is easy to integrate into a host cell genome and destroy the sequence in the genome, an episomal CRISPR/Cas9 expression vector is provided, which is a CRISPR/Cas9 expression vector containing an S/MAR sequence, wherein the nucleotide of the S/MAR sequence is shown as SEQ ID NO:1 is shown in the specification; the S/MAR sequence is located between the coding region sequence capable of coding for a protein and the bGH poly (A) sequence in this vector.

Further defined, the sequence of the coding region capable of coding the protein is the sequence of the EGFP coding region.

Further defined, the sgRNA sequence of the CRISPR/Cas9 expression vector is SEQ ID NO: 2-4.

Further defined, the episomal CRISPR/Cas9 expression vector further comprises a G418 resistance gene, the nucleotide sequence of which is set forth in SEQ ID NO:5, respectively.

The invention also provides a construction method of the addition type CRISPR/Cas9 expression vector, which constructs an S/MAR sequence between a coding region sequence capable of coding protein and a bGH poly (A) sequence of the CRISPR/Cas9 expression vector by an enzyme digestion connection or homologous recombination method.

Further defined, the method for constructing the episomal CRISPR/Cas9 expression vector comprises the following steps:

1) Constructing a G418 resistance gene into a px330-EGFP vector to obtain a vector skeleton px330-EGFP-G418; the nucleotide sequence of the G418 resistance gene is shown as SEQ ID NO:5 is shown in the specification;

2) Constructing an S/MAR sequence on the vector skeleton px330-EGFP-G418 obtained in the step 1) to obtain a vector skeleton px330-EGFP-G418-S/MAR;

3) Inserting an sgRNA sequence into a BbsI enzyme cutting site behind a U6 promoter of a vector px330-EGFP-G418-S/MAR expression skeleton to obtain an additional CRISPR/Cas9 expression vector px330-EGFP-G418-S/MAR-sgRNA, wherein the sgRNA sequence is shown in SEQ ID NO: 2-4.

Further limiting, the G418 resistance gene and the px330-EGFP vector in the step 1) are respectively cut by BsmBI enzyme, and the px330-EGFP vector framework enzyme cutting large fragment and G4 are recovered18 resistant gene enzyme cutting product, by T ₄ DNA ligase to obtain vector skeleton px330-EGFP-G418.

Further limiting, the S/MAR sequence and the px330-EGFP-G418 vector in the step 2) are respectively cut by Bsp1407, the cut large fragment and the S/MAR sequence of the px330-EGFP-G418 vector backbone are recovered and are subjected to T ₄ And performing DNA enzyme connection to obtain a vector skeleton px330-EGFP-G418-S/MAR.

Further limiting, the sgRNA sequence and the vector skeleton px330-EGFP-G418-S/MAR in the step 3) are respectively subjected to BbsI enzyme digestion, the large enzyme digestion fragment and the sgRNA sequence digestion product of the vector skeleton px330-EGFP-G418-S/MAR are recovered, and the product is subjected to T enzyme digestion ₄ And (3) performing DNA enzyme connection to obtain an additional CRISPR/Cas9 expression vector px330-EGFP-G418-S/MAR-sgRNA.

The addition type CRISPR/Cas9 expression vector can be used for gene knockout research.

Advantageous effects

Matrix Attachment Regions (MARs) refer to DNA sequences in eukaryotic chromatin that are capable of specifically binding to the matrix (nuclear) or the nuclear backbone, also known as nuclear backbone attachment regions (SAR). In the present invention, the S/MAR (or SMAR) sequence is a naked DNA sequence in a chromosome, and is mainly combined with a nuclear matrix or a nuclear skeleton in a cell nucleus to stabilize the spatial position of the chromosome. After the S/MAR sequence is connected with the CRISPR/Cas9 plasmid system, the plasmid vector constructed by the CRISPR/Cas9 and the S/MAR can exist outside a chromosome in a circular stable free manner, and compared with the conventional CRISPR/Cas9 viral vector and plasmid vector, the CRISPR/Cas9 viral vector and plasmid vector do not need to be integrated into a host cell genome, so that the sequence in the genome cannot be damaged, and the vector has high safety and small potential safety hazard.

Meanwhile, the S/MAR can enable the CRISPR/Cas9 expression vector to form an independent ring structure outside a chromosome, so that gene silencing caused by gene position effect in the chromosome is avoided. In view of this relationship between S/MAR sequences and gene expression, in particular it overcomes positional effects, avoiding transgene silencing, and has been applied in transgenic bioengineering as a cis-regulatory element. By constructing a proper expression vector, connecting S/MAR on one side of the target gene and then introducing the target gene into animal cells, the target gene can be stably expressed, and the difference of the expression level of the exogenous gene among transgenic individuals is reduced.

The episomal CRISPR/Cas9 expression vector provided by the invention can be used for dissociating and expressing Cas9 protein and single-stranded guide RNA outside a chromosome, and avoids exogenous gene from being integrated into a host cell genome, so that the sequence in the genome is damaged, and potential safety hazards are effectively avoided.

Drawings

FIG. 1 is a PCR amplification G418 sequence electrophoresis chart, in which M is DNA Marker8000, and 1-3 are G418 sequences;

FIG. 2 is a sequence electrophoretogram of PCR amplified S/MAR, in which M is DNA Marker8000 and 1-2 are SMAR sequences;

FIG. 3 is a graph of the cleavage electrophoresis of a G418 and S/MAR sequence vector, wherein M is DNA Marker15000,1 is a graph of the cleavage electrophoresis result of vector px330-EGFP-G418 by Bsp1407, and 2 is a graph of the cleavage electrophoresis result of vector px330-EGFP-G418-S/MAR by BglII;

FIG. 4 is a diagram showing the results of annealing electrophoresis of sgRNA, wherein M is DNA Marker15000,1 is a sequence of the sgRNA of the CYP20A1 gene, 2 is a sequence of the sgRNA of the SIRT3 gene, and 3 is a sequence of the sgRNA of the ANXA7 gene;

FIG. 5 is a BglII enzyme digestion electrophoresis result diagram of the constructed vector, wherein M is DNA Marker15000,1 is a BglII enzyme digestion electrophoresis result diagram of a CYP20A1 gene targeting vector, 2 is a BglII enzyme digestion electrophoresis result diagram of a SIRT3 gene vector, and 3 is a BglII enzyme digestion electrophoresis result diagram of an ANXA7 gene targeting vector;

FIG. 6 schematic representation of the targeting vector px 330-EGFP-G418-S/MAR-sgRNA;

FIG. 7 positive cell screening results;

FIG. 8 shows the electrophoresis of the plasmid restriction enzyme extracted from the cell, wherein M is DNA Marker15000,1-2 are the electrophoresis results of the CYP20A1 gene targeting vector after Bsp1407 restriction enzyme digestion, and 3 is the electrophoresis results of the CYP20A1 gene targeting vector.

Detailed Description

Description of the terms or abbreviations:

g418 resistance gene: a neomycin resistance gene;

S/MAR is a nuclear matrix attachment region or a nuclear framework attachment region;

sgRNA: small guide RNA of small guide RNA;

PBS: phosphate buffer.

Enzyme digestion connection: the method is characterized in that two or more than two DNA fragments which are cut by restriction endonuclease are connected by DNA ligase.

Homologous recombination: it is meant that two or more DNA fragments having 20-25bp repetitive sequences at their ends are ligated by a DNA recombinase.

The invention is characterized in that:

the px330-EGFP vector was purchased from addgene, cat # 66581, from which the EGFP coding region sequence, bGH poly (a) sequence, U6 promoter sequence described herein were derived;

the pEGFP-C1 vector was purchased from Clontech, cat # 6084-1;

pEI-1 vectors are described in Stefano Manzini, alessia Vargiolu, isa M Stehle, maria Laura Bacci, maria Grazia Cerrito, roberto Giovannoni, augusta Zannoni, maria Rosa Bianco, monica Forni, pierluigi Donini, michele Papa, hans J Lipps, and Mariala Laviar.Genetic modified pigs produced with a nonviral isolated vector, PNAS November 21,2006 (47) 17672-17677; https:// doi.org/10.1073/pnas.0604938103, publicly available through northeast university of agriculture.

Porcine fibroblasts: the pig primary fibroblast is obtained by culturing pig primary fibroblasts, and the culturing method is a conventional method;

serum-free and double-antibody-free DMEM culture solution: DMEM medium (cat # C11885500 BT) from Gibco;

DMEM medium with 10% serum: DMEM culture solution added with 10% fetal calf serum and double antibodies;

the selection medium digestive juice (0.25% of trypsin) refers to digestive juice with the mass fraction of trypsin being 0.25%;

other reagents or instrumentation, unless otherwise specified, are commercially available.

Example 1 episomal CRISPR/Cas9 expression vector.

The episomal CRISPR/Cas9 expression vector described in this example refers to a CRISPR/Cas9 expression vector containing S/MAR sequence whose nucleotides are set forth in SEQ ID NO:1 is shown in the specification; the S/MAR sequence is located between the coding region sequence capable of coding for a protein and the bGH poly (A) sequence in this vector.

Example 2 episomal CRISPR/Cas9 expression vectors.

The episomal CRISPR/Cas9 expression vector described in this example refers to a CRISPR/Cas9 expression vector containing S/MAR sequence whose nucleotides are set forth in SEQ ID NO:1, the CRISPR/Cas9 expression vector contains an EGFP coding region sequence, such as an EGFP coding region sequence from a px330-EGFP vector, and the S/MAR sequence is located between the EGFP coding region sequence and a bGH poly (a) sequence in the CRISPR/Cas9 expression vector.

Example 3 episomal CRISPR/Cas9 expression vectors.

The episomal CRISPR/Cas9 expression vector of this embodiment refers to a CRISPR/Cas9 expression vector comprising an S/MAR sequence whose nucleotides are set forth in SEQ ID NO:1 is shown in the specification; the S/MAR sequence is positioned between a coding region sequence capable of coding protein and a bGH poly (A) sequence in the vector, and the sgRNA sequence in the CRISPR/Cas9 expression vector is SEQ ID NO: 2-4.

Example 4 episomal CRISPR/Cas9 expression vectors.

The episomal CRISPR/Cas9 expression vector described in this example refers to a CRISPR/Cas9 expression vector containing S/MAR sequence whose nucleotides are set forth in SEQ ID NO:1 is shown in the specification; the S/MAR sequence is located between the coding region sequence capable of coding for a protein and the bGH poly (A) sequence in the vector.

To further analyze targeting efficiency, the episomal CRISPR/Cas9 expression vector described in this example further contains a G418 resistance gene, the nucleotide sequence of which is set forth in SEQ ID NO:5, respectively.

Example 5. Construction method of episomal CRISPR/Cas9 expression vector.

The construction method of the addition type CRISPR/Cas9 expression vector described in the embodiment refers to that an SMAR sequence is constructed between a coding region sequence capable of coding a protein and a bGH poly (A) sequence in the CRISPR/Cas9 expression vector by an enzyme digestion connection or homologous recombination method.

The following examples are described below:

1. constructing a G418 resistance gene into a px330-EGFP vector to obtain a vector skeleton px330-EGFP-G418; the nucleotide sequence of the G418 resistance gene is shown as SEQ ID NO:5 is shown in the specification; the method comprises the following specific steps:

1) G418 resistance gene sequence was obtained.

And designing a primer according to the G418 resistance gene sequence in the pEGFP-C1 vector for PCR amplification to obtain the G418 resistance gene sequence.

The PCR reaction system was a 50. Mu.L system (TaKaRa, RR 902): ex Taq 25. Mu.L; an upstream primer Cas 9-Kana-F1 mu L; a downstream primer Cas 9-Kana-R1 mu L; total DNA template 1. Mu.L (10 ng); ddH ₂ O22 mu L; the PCR reaction conditions were: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s for 33 cycles, and extension at 72 ℃ for 10min.

The PCR products of the G418 resistance genes were each verified by electrophoresis, and as shown in FIG. 1, agarose gel DNA recovery was carried out using the TIANGel Midipurification Kit (TIANGEN DP 209-03) of TIANGEN corporation. The specific steps are carried out according to the operation of the kit.

TABLE 1 vector construction primers

2) BsmBI digested the G418 resistance gene and the px330-EGFP plasmid vector.

The px330-EGFP plasmid vector and the G418 resistance gene fragment DNA were cut with BsmBI (NEB, R0134L) in a 10. Mu.L system: bsmBI enzyme 1 uL; 1 μ L of 10xBuffer; plasmid DNA (or G418 resistance gene fragment) 5. Mu.L (200 ng); ddH ₂ O3. Mu.L. The digestion was carried out at the optimum reaction temperature of BsmBI enzyme of 55 ℃ for 8 h. And (3) detecting the enzyme digestion result by electrophoresis, and recovering the G418 resistance gene and the px330-EGFP vector framework large fragment from the enzyme digestion fragment by using an agarose gel DNA kit.

3) The G418 resistance gene and the px330-EGFP vector were ligated.

T ₄ Ligase (NEB, M0202L) linker was a 10 μ L system: 1 mu L of px330-EGFP enzyme digestion product; 7 mu L of G418 resistance gene enzyme digestion product; t is ₄ 1 mu L of ligase; solutionI 1 μ L. The ligation system was mixed and ligated overnight at 16 ℃ to form a new vector backbone px330-EGFP-G418, DH5 α competent bacteria were transformed the next day and ligation products were amplified in large quantities, and plasmid miniprep was performed using a plasmid miniprep kit (TIANGEN, DP 103-03) to obtain a high concentration vector backbone. Sent to Shanghai Yingjun sequencing company for sequencing verification using G418 sequencing primer.

TABLE 2 sequencing primers

2. Constructing an S/MAR sequence on the vector skeleton px330-EGFP-G418 obtained in the step 1 to obtain a vector skeleton px330-EGFP-G418-S/MAR; the method comprises the following specific steps:

1) S/MAR sequence acquisition

And designing a primer according to the S/MAR sequence in the pEI-1 vector for PCR amplification to obtain the S/MAR sequence. PCR reaction system (TaKaRa, RR 902)

50 μ L system: ex Taq 25. Mu.L; an upstream primer Cas 9-SMAR-F1 mu L; a downstream primer Cas 9-SMAR-R1 mu L; total DNA template 1 μ L (10 ng);

ddH ₂ o22 mu L; the PCR reaction conditions are as follows: 94 ℃ 5min,94 ℃ 30s,60 ℃ 45s,72 ℃ 1min40s for a total of 33 cycles, followed by extension at 72 ℃ for 10min.

The post-PCR electrophoresis of the S/MAR was verified, and as shown in FIG. 2, the product recovery was performed using agarose gel DNA recovery kit (TIANGEN DP 209-03), and the specific experimental steps were performed according to the kit instructions.

TABLE 3 vector construction primers

2) Bsp1407 cuts the S/MAR sequence and the px330-EGFP-G418 vector framework.

The px330-EGFP-G418 plasmid vector and the S/MAR sequence DNA are respectively cut by Bsp1407, and the cutting system is a 20 mu L system: bsp 1407. Mu.L; 10xLoading Buffer 2 μ L; DNA fragment (plasmid) 8. Mu.L (200 ng); ddH ₂ O8. Mu.L. The enzyme digestion system is mixed and then is digested for 3 hours at 37 ℃.

3) The S/MAR is linked to the px330-EGFP-G418 backbone.

The linker is a 10 μ L system: 1 mu L of enzyme digestion product of px330-EGFP-G418; 7 mu L of S/MAR enzyme digestion product; t is ₄ Ligase 1. Mu.L (NEB M0202L); solutionI 1 μ L. And mixing the connection systems, connecting for 8h at 16 ℃ to form a new vector skeleton px330-EGFP-G418-S/MAR, transforming DH5 alpha competent cells on the next day, amplifying a large amount of connection products, extracting a small amount of plasmids, and generating a vector through DH5 alpha amplification, wherein the vector skeleton is preliminarily constructed and stored in a refrigerator at 4 ℃ for later use.

TABLE 4 sequencing primers

3. Constructing a sgRNA sequence into a vector skeleton px330-EGFP-G418-SMAR, and performing BbsI enzyme cutting site behind a U6 promoter to obtain an additional CRISPR/Cas9 expression vector px330-EGFP-G418-S/MAR-sgRNA, wherein the sgRNA sequence is shown in SEQ ID NO: 2-4. The method comprises the following specific steps:

1) sgRNA sequence design

sgRNA sequences were designed. The targeting sites were designed from the exon sequences of the MC4R coding region of swine using the website (http;/crispr. Mit. Edu /) according to the design rules of sgRNA. The sgRNA has two different regions, and the total length is about 80 bp. Firstly, a base sequence of about 20bp which can be complementary with a target DNA exists at the 5' end of the target DNA, a hairpin structure is formed through annealing processing, and the Cas9 protein is guided to perform gene knockout on a target site of 200 bases in front of a CDS region of the target DNA for cutting. Meanwhile, a PAM structure (PAM) which is combined with a fragment containing two guanine residues (-NGG) or a guanine residue and an adenine residue (-NAG) and contains two cytosine residues (-NCC) exists on the sgRNA, and then a sequence with low off-target rate is selected and synthesized according to the sequences given on the network to design the sgRNA sequence. The target genes are respectively: cytochrome P450, family 20, subfamily A, polypeptide 1 (CYP 20A 1), NAD-dependent deacetylase sirtuin-3 (SIRT 3) and Annexin A7 (ANXA 7) genes.

TABLE 5sgRNA sequences

According to the designed sgRNA sequences, 2 single-stranded DNA sequences are respectively designed to be complementary single strands of a sgRNA sense strand and an antisense strand, wherein the two ends of the two complementary strands are provided with BbsI enzyme cutting sites. The sense strand is a restriction enzyme site (BbsI), a sgRNA sequence and a restriction enzyme site (BbsI) in the order of 5 '-3'. The antisense strand is a cleavage site (BbsI), an antisense sgRNA sequence, and a cleavage site (BbsI) in the order of 3 '-5'. And then the two strands are annealed to form a complementary DNA double strand, the two ends of the complementary DNA double strand are BbsI enzyme cutting sites, and the complementary DNA double strand can be directly connected with px330-EGFP-G418-S/MAR after BbsI enzyme cutting. CYP20A1-gRNA-1-F1 and CYP20A1-gRNA-1-R1 respectively; SIRT3-gRNA-2-F1, SIRT3gRNA-2-R1; ANXA7-gRNA-3-F1 and ANXA7-gRNA-3-R1. Each group of single strands forms double strands with hairpin structures after annealing at 55 ℃, and the electrophoresis detection result is shown in figure 4.

Table 6 sgRNA sequences after synthesis of double strands

TABLE 7 sequencing primers

2) Targeting vector construction

The targeting vector frameworks px330-EGFP-G418-SMAR and sgRNA are subjected to single enzyme digestion by BbsI (NEB company, R0539V), and products after enzyme digestion are connected after being recovered by glue to form complete circular recombinant plasmids.

(1) And (3) carrying out BbsI enzyme digestion on the px330-EGFP-G418-SMAR and the sgRNA.

The enzyme cutting system is a 50 mu L system: bsmbI enzyme 5. Mu.L; NE Buffer 2.1 u L; plasmid vector (or sgRNA) 20. Mu.L (200 ng). The resulting mixture was placed in a PCR apparatus at 37 ℃ overnight for digestion. Electrophoresis and recovery of the enzyme digestion product glue.

(2) And px330-EGFP-G418-SMAR is connected with gRNA.

The linker was 10 μ L: plasmid vector 1. Mu.L (10 ng); sgRNA cleavage product 7. Mu.L (100 ng); t is ₄ 1 μ L of Ligase; solutionI 1 μ L. Placing in a constant temperature water bath kettle, connecting overnight at 16 ℃ to obtain a targeting vector px330-EGFP-G418-S/MAR-sgRNA, wherein the vector map is shown in figure 6.

(3) The recombinant plasmid px330-EGFP-G418-S/MAR-sgRNA was transformed into DH5 α.

The temperature of the constant-temperature water bath is adjusted to 42 ℃, an ultra-clean bench ultraviolet lamp is turned on for 30min, a tube (100 mu L) of the allelochemicals is taken out from a refrigerator at minus 80 ℃, and the ice bath is carried out for 5-10min until the allelochemicals is in a molten state. Adding 10 mu L of the connected plasmid mixed solution into a molten state of competent bacteria, slightly shaking, carrying out ice bath for 30min, taking out ice bath competent cells, carrying out water bath at 42 ℃ for 45s, then quickly putting back on ice, carrying out ice bath for 2min, adding 500 mu L of SOC culture solution (without antibiotics) into the tube on a super clean bench, slightly mixing, fixing on a constant temperature shaking bed at 37 ℃, shaking at 200rpm for 1h, taking 500 mu L of clear solution, uniformly and flatly coating on an SOC culture medium, and placing in a constant temperature box at 37 ℃ for overnight culture.

(4) And (4) plasmid extraction.

The specific experimental operation steps of extracting Plasmid (TIANGEN, DP 103-03) by using the TIANGEP Mini Plasmid Kit of the small Plasmid extraction Kit of the Tiangen are carried out according to the Kit operation instructions. And obtaining positive plasmids through enzyme digestion sequencing identification.

1. And (3) inspecting the targeting effect and the dissociative verification of the addition type CRISPR/Cas9 expression vector.

1. Cell recovery

And taking out the cryopreserved pig fibroblast cryopreserving tube from the liquid nitrogen tank, and quickly putting the cryopreserved pig fibroblast cryopreserving tube into a water bath kettle at the temperature of 37-40 ℃ for quick dissolution. Adding appropriate amount of DMEM culture solution containing 10% serum into the cell precipitate, gently blowing, mixing, transferring the cell suspension into a culture dish, replenishing the culture solution, adding 5% CO at 37 deg.C ₂ Culturing in an incubator, continuously culturing by changing culture solution after thawing for 24h, and evaluating the cryopreservation efficiency according to the proportion of adherent cells after the cells are recovered for 24 h.

2. Subculturing of cells

(1) Cleaning and sterilizing required culture tools, placing the culture tools into a clean bench, performing ultraviolet sterilization for 30min, taking out the culture bottles with the formed compact monolayer cells from the incubator, placing the culture bottles into the clean bench, igniting an alcohol lamp, and pouring culture solution beside the alcohol lamp into a small beaker.

(2) Add 500. Mu.L of digest (0.25% trypsin) to the flask, shake the flask gently to wet the whole cell monolayer with the digest, and leave it at room temperature for 2-3min. The flask was turned over with the bottom facing down and the cell monolayer was visualized with the naked eye and the digestive juices were decanted off when a void (about pinhole size) appeared in the monolayer.

(3) Adding 3.5mL of culture solution into the culture flask to stop the digestion of trypsin, sucking the culture solution in the flask by using a suction pipe, repeatedly impacting cells on the flask wall until all the cells are flushed down, and gently mixing to prepare a cell suspension.

(4) Aspirating a small amount of cell suspension into the chamber of a cell counter, counting under a light microscope, and adjusting the cell concentration to 5X 10 per ml according to the result ⁵ And (4) one cell.

(5) 2mL of the cell suspension was aspirated and transferred to another flask, and 2mL of the cell suspension was left in the original flask (the rest was discarded), and 4mL of a fresh culture solution was added to each flask, capped with a bottle stopper, and the flask was gently shaken and then placed in a 37 ℃ incubator for culture.

3. Liposome transfection

Liposome transfection was performed according to kit instructions (Shanghai Boyao Bio 11668-019 1.5mL) and transfection was prepared when the cells grew to 75% density. And (2) adding 100 mu L of serum-free and double-antibody-free DMEM culture solution into each of 2 EP tubes, adding 4 mu G of px330-EGFP-G418-S/MAR-sgRNA plasmid into the tube A, adding 8 mu L of liposome into the tube B, and incubating for 5min. Then, the liquid in the tube A and the tube B is mixed, the mixture is blown and beaten uniformly by a pipette gun, and the complex is incubated for 15min at 37 ℃. And (3) taking cells to be transfected, discarding old culture solution, washing the cells twice by using serum-free and double-antibody-free DMEM culture solution, and supplementing 1.8mL of serum-free and double-antibody-free DMEM into each hole of a 6-hole culture plate. Slowly adding the A/B compound into a serum-free and double-antibody-free DMEM culture medium, gently shaking the cell culture plate while dripping, placing in an incubator at 37 ℃ for 8 hours, and replacing 2mL of DMEM culture solution containing 10% serum to continue culturing.

4. Screening of target cells

Drug screening targeting cells: the following day of cell transfection, the ratio of 1:10 (volume ratio) passage into new culture dish overnight culture, then PBS washing and change the screening medium, according to the concentration of the pre-experiment groping add G418 (Sigma), the concentration is 1000ng/mL, control group cells for not transfection of carrier plasmid, but add G418 drug. After the control group cells died after 4 days, the test group cells were replaced with the selection medium (G418 concentration 200 ng/mL). And (4) cleaning with sterile PBS solution every 1 day, replacing a screening culture medium, and performing monoclonal picking after the cells grow enough in about five days.

5. Verification of targeting efficiency

(1) And extracting total DNA of adherent cells.

Total DNA of adherent cells was extracted with the Genomic DNA Extraction Kit TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (TaKaRa 9765). The details are as follows (all reagents used are from the kit):

discarding the culture medium to 10cm ² Adding 1ml of LPBS into the adherent cells, blowing off the adherent cells by using a gun head of a liquid transfer gun, transferring the adherent cells into a new 1.5ml of LEP tube, centrifuging at 12000rpm for 1min, sucking and removing supernatant, adding 10 mu L of deionized water, and blowing and beating uniformly by using the gun. Adding 180 mu.L of BufferGL, 20 mu.L of ProteinaseK and 10 mu.L of RNaseA9 (10 mg/mL), carrying out water bath at 56 ℃ until the tissues are completely cracked, centrifuging at 12000rpm for 2min after cracking, and removing impurities and then carrying out subsequent operations. Towards the crackingAdd 200. Mu.L of LBufferGB and 200. Mu.L of 100% ethanol to the solution, suck well and mix well. The Spin Column was mounted on the Collection Tube, the solution was transferred to the Spin Column and centrifuged at 12000rpm for 2min. mu.L of BufferWA was added to Spin Column, centrifuged at 12000rpm for 1min, and the filtrate was discarded. mu.L of BufferWB (containing ethanol) was added to Spin Column, centrifuged at 12000rpm for 1min, and the filtrate was discarded and repeated once. Spin columns were mounted on the Collection Tube and centrifuged at 12000rpm for 2min. Spin Column was placed in a new 1.5mL centrifuge tube, 100 μ L of deionized water was added to the center of the Spin Column membrane, and allowed to stand at room temperature for 5min. DNA was eluted by centrifugation at 12000rpm for 2min. The spectrophotometer quantitated and the samples were placed in a 4 ℃ display cabinet for storage.

(2) CYP20A1, SIRT3 and ANXA7 targeting sequences are amplified by PCR.

Primers aiming at exons of CYP20A1, SIRT3 and ANXA7 coding regions of pigs are used for amplification, sequence information is obtained after PCR sequencing, and the sequence information is compared with an original sequence to check the targeting efficiency.

TABLE 8 Gene DNA sequence amplification primers

TABLE 9 sequencing results of CYP20A1 Gene-targeted genomic DNA

Original sequence CYP20A1	5’-CTTCCACTGTTCACAATATCTGG-3’
		Mutant sequence 1	5’-CTTCCACTGTTCACAAT-TCTGG-3’
Mutant sequence 2	5’-CTTCCACTGTTCACAA--TCTGG-3’
		Mutant sequence 3	5’-CTTCCACTGTTCAC------TGG-3’
Mutant sequence 4	5’-CTTCCACTGTTCACAA----TGG-3’

TABLE 10SIRT3 Targeted genomic DNA sequencing results

Original sequence SIRT3	5’-AAGCCCGACATCGTGTTCTT TGG-3’
		Mutant sequence 1	5’-AAGCCCGACATCGT-TTCTT TGG-3’
Mutant sequence 2	5’-AAGCCCGACATCG--TTCTT TGG-3’
		Mutant sequence 3	5’-AAGCCCGACA----GTTCTT TGG-3’
Mutant sequence 4	5’-AAGCCCGACA---TGTTCTT TGG-3’

TABLE 11ANXA7 Targeted genomic DNA sequencing results

Original sequence ANXA7	5’-GGTCAGTATCCTTATCCTAG TGG-3’
		Mutant sequence 1	5’-GGTCAGTATCCTTA-CCTAG TGG-3’
Mutant sequence 2	5’-GGTCAGTATCCT---CCTAG TGG-3’
		Mutant sequence 3	5’-GGTCAGTAT-----TCCTAG TGG-3’
Mutant sequence 4	5’-GGTCAGTAT---TATCCTAG TGG-3’

The targeting results are set forth in the following table: sequencing results show that mutations are generated at target sites of sgRNAs, and the CRISPR/Cas9 vector system is proved to normally function in cells.

6. Verification of vector liberation

(1) Plasmid DNA extraction from cells.

After the prepared px330-EGFP-G418-SMAR-sgRNA (targeting CYP20A1 gene) transfects pig fibroblasts, monoclonals are picked after cell screening in the step 4, and related operations are carried out according to the operation instruction of a adenovirus extraction Kit Vader-TrpTMHigh Purity miniSpin Plasmid Purification Kit (VIRONGY NO. VR8880628), and vector plasmids existing in cells in a free mode are extracted.

(2) And (4) transformation.

The temperature of the constant-temperature water bath is adjusted to 42 ℃, an ultra-clean bench ultraviolet lamp is turned on for 30min, a tube (100 mu L) of DH5 alpha allelopathic bacteria is taken out from a refrigerator at minus 80 ℃, and the ice bath is carried out for 5-10min until the bacteria is in a molten state. Adding 10 mu L of the extract in the previous step into a molten allelochemicals, slightly shaking, carrying out ice bath for 30min, taking out ice-bath competent cells, carrying out water bath at 42 ℃ for 45s, then rapidly returning to ice, carrying out ice bath for 2min, adding 500 mu LSOC culture solution (without antibiotics) into the tube on a super clean bench, slightly mixing, fixing on a constant temperature shaking bed at 37 ℃, shaking at 200rpm for 1h, taking 500 mu L of clear liquid, uniformly and flatly coating on an SOC culture medium, and placing in a constant temperature box at 37 ℃ for overnight culture.

(3) And (4) plasmid extraction.

And (3) carrying out mass amplification culture on the positive clone bacteria obtained in the last step. The specific experimental operation steps of extracting Plasmid (TIANGEN, DP 103-03) by using a small Plasmid extraction Kit TIAN prep Mini Plasmid Kit of the Tiangen are carried out according to the Kit operation instructions.

(4) The extracted plasmid was identified by digestion with Bsp1407 endonuclease as shown in FIG. 8, and the plasmid was sent to Shanghai Yingjun sequencing company for sequencing verification using G418 sequencing primer.

The results show that: the circular plasmid vector exists in the pig fibroblast screened by neomycin, and as shown in figure 8, the restriction enzyme digestion identification result shows that the size of the plasmid is consistent with the expected size by taking the targeting vector of the CYP20A1 gene as an example. The sequencing result shows that the plasmid is a px330-EGFP-G418-S/MAR-sgRNA (targeted CYP20A1 gene) vector. These results indicate that circular px330-EGFP-G418-SMAR-sgRNA (targeted CYP20A1 gene) plasmid vector still exists in pig fibroblasts screened for a long time by neomycin, indicating that S/MAR can make the plasmid vector exist stably extrachromosomally.

The other two targeting vectors aiming at the target genes SIRT3 and ANXA7 respectively have similar results of vector free verification to the results of the CYP20A1 targeting vector, and the results also prove that the S/MAR can ensure that the plasmid vector exists stably outside the chromosome.

Nucleotide sequence listing

<110> northeast university of agriculture

<120> additional CRISPR/Cas9 expression vector and construction method and application thereof

<130>

<160> 39

<170> PatentIn version 3.5

<210> 1

<211> 1995

<212> DNA

<213> S/MAR sequences

<400> 1

agatctaaat aaacttataa attgtgagag aaattaatga atgtctaagt taatgcagaa 60

acggagagac atactatatt catgaactaa aagacttaat attgtgaagg tatactttct 120

tttcacataa atttgtagtc aatatgttca ccccaaaaaa gctgtttgtt aacttgtcaa 180

cctcatttca aaatgtatat agaaagccca aagacaataa caaaaatatt cttgtagaac 240

aaaatgggaa agaatgttcc actaaatatc aagatttaga gcaaagcatg agatgtgtgg 300

ggatagacag tgaggctgat aaaaagagta gagctcagaa acagacccat tgatatatgt 360

aagtgaccta tgaaaaaaat atggcatttt acaatgggaa aatgatgatc tttttctttt 420

ttagaaaaac agggaaatat atttatatgt aaaaaataaa agggaaccca tatgtcatac 480

catacacaca aaaaaattcc agtgaattat aagtctaaat ggagaaggca aaactttaaa 540

tcttttagaa aataatatag aagcatgcca tcatgacttc agtgtagaga aaaatttctt 600

atgactcaaa gtcctaacca caaagaaaag attgttaatt agattgcatg aatattaaga 660

cttattttta aaattaaaaa accattaaga aaagtcaggc catagaatga cagaaaatat 720

ttgcaacacc ccagtaaaga gaattgtaat atgcagatta taaaaagaag tcttacaaat 780

cagtaaaaaa taaaactaga caaaaatttg aacagatgaa agagaaactc taaataatca 840

ttacacatga gaaactcaat ctcagaaatc agagaactat cattgcatat acactaaatt 900

agagaaatat taaaaggcta agtaacatct gtggcaatat tgatggtata taaccttgat 960

atgatgtgat gagaacagta ctttacccca tgggcttcct ccccaaaccc ttaccccagt 1020

ataaatcatg acaaatatac tttaaaaacc attaccctat atctaaccag tactcctcaa 1080

aactgtcaag gtcatcaaaa ataagaaaag tctgaggaac tgtcaaaact aagaggaacc 1140

caaggagaca tgagaattat atgtaatgtg gcattctgaa tgagatccca gaacagaaaa 1200

agaacagtag ctaaaaaact aatgaaatat aaataaagtt tgaactttag ttttttttaa 1260

aaaagagtag cattaacacg gcaaagtcat tttcatattt ttcttgaaca ttaagtacaa 1320

gtctataatt aaaaattttt taaatgtagt ctggaacatt gccagaaaca gaagtacagc 1380

agctatctgt gctgtcgcct aactatccat agctgattgg tctaaaatga gatacatcaa 1440

cgctcctcca tgttttttgt tttcttttta aatgaaaaac tttatttttt aagaggagtt 1500

tcaggttcat agcaaaattg agaggaaggt acattcaagc tgaggaagtt ttcctctatt 1560

cctagtttac tgagagattg catcatgaat gggtgttaaa ttttgtcaaa tgctttttct 1620

gtgtctatca atatgaccat gtgattttct tctttaacct gttgatggga caaattacgt 1680

taattgattt tcaaacgttg aaccaccctt acatatctgg aataaattct acttggttgt 1740

ggtgtatatt ttttgataca ttcttggatt ctttttgcta atattttgtt gaaaatgttt 1800

gtatctttgt tcatgagaga tattggtctg ttgttttctt ttcttgtaat gtcattttct 1860

agttccggta ttaaggtaat gctggcctag ttgaatgatt taggaagtat tccctctgct 1920

tctgtgttct gaaagagatt gtagaaagtt gatacaattt ttttttcttt aaatatcttg 1980

atagaattct gtaca 1995

<210> 2

<211> 23

<212> DNA

<213> sgRNA-CYP20A1

<400> 2

cttccactgt tcacaatatc tgg 23

<210> 3

<211> 23

<212> DNA

<213> sgRNA-SIRT3

<400> 3

aagcccgaca tcgtgttctt tgg 23

<210> 4

<211> 23

<212> DNA

<213> sgRNA-ANXA7

<400> 4

ggtcagtatc cttatcctag tgg 23

<210> 5

<211> 1771

<212> DNA

<213> G418 resistance Gene

<400> 5

tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 60

gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga 120

aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca 180

ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca 240

ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc 300

ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc 360

catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta 420

ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga 480

gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc 540

cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga 600

tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct 660

gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac 720

gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct 780

attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt 840

atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt 900

cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt 960

cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag 1020

gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt 1080

gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg 1140

tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg 1200

cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg 1260

catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg 1320

accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat 1380

gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 1440

gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag 1500

acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt 1560

gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc 1620

ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc 1680

ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata 1740

gcctcaggtt actcatatat actttagatt g 1771

<210> 6

<211> 33

<212> DNA

<213> Cas9-Kana-F

<400> 6

ggcgtctccg ggtgtgcgcg gaacccctat ttg 33

<210> 7

<211> 38

<212> DNA

<213> Cas9-Kana-R

<400> 7

ggcgtctcgc gcgcaatcta aagtatatat gagtaacc 38

<210> 8

<211> 25

<212> DNA

<213> G418 primer for sequencing Cas9-G418-F

<400> 8

acccgctgac gcgccctgac gggct 25

<210> 9

<211> 33

<212> DNA

<213> Cas9-SMAR-F

<400> 9

gctgtacaag atctaaataa acttataaat tgt 33

<210> 10

<211> 33

<212> DNA

<213> Cas9-SMAR-R

<400> 10

gctgtacaga attctatcaa gatatttaaa gaa 33

<210> 11

<211> 28

<212> DNA

<213> SMAR sequencing primer-Cas 9-SMAR-R

<400> 11

ttattaggaa aggacagtgg gagtggca 28

<210> 12

<211> 24

<212> DNA

<213> CYP20A1-gRNA-1-F1

<400> 12

cacccttcca ctgttcacaa tatc 24

<210> 13

<211> 24

<212> DNA

<213> CYP20A1-gRNA-1-R1

<400> 13

aaacgatatt gtgaacagtg gaag 24

<210> 14

<211> 24

<212> DNA

<213> SIRT3-gRNA-2-F1

<400> 14

caccaagccc gacatcgtgt tctt 24

<210> 15

<211> 24

<212> DNA

<213> SIRT3-gRNA-2-R1

<400> 15

aaacaagaac acgatgtcgg gctt 24

<210> 16

<211> 24

<212> DNA

<213> ANXA7-gRNA-3-F1

<400> 16

caccggtcag tatccttatc ctag 24

<210> 17

<211> 24

<212> DNA

<213> ANXA7-gRNA-3-R1

<400> 17

aaacctagga taaggatact gacc 24

<210> 18

<211> 25

<212> DNA

<213> primer Cas9-SgRNA-F for SgRNA sequencing

<400> 18

ctgttagaga gataattgga attaa 25

<210> 19

<211> 24

<212> DNA

<213> ANAX7-S

<400> 19

ttagaaggat tatgtttctc aatg 24

<210> 20

<211> 22

<212> DNA

<213> ANAX7-A

<400> 20

gaagtggaaa gtagcctaag tg 22

<210> 21

<211> 18

<212> DNA

<213> CYP20A1-S

<400> 21

acagctagtt cagaatca 18

<210> 22

<211> 17

<212> DNA

<213> CYP20A1-A

<400> 22

tgtatggtct tcctttt 17

<210> 23

<211> 21

<212> DNA

<213> SIRT3-S

<400> 23

gagttttccc ttttccccta a 21

<210> 24

<211> 15

<212> DNA

<213> SIRT3-A

<400> 24

agccccgcct gcttt 15

<210> 25

<211> 23

<212> DNA

<213> original sequence CYP20A1

<400> 25

cttccactgt tcacaatatc tgg 23

<210> 26

<211> 22

<212> DNA

<213> CYP20A 1-mutant sequence 1

<400> 26

cttccactgt tcacaattct gg 22

<210> 27

<211> 21

<212> DNA

<213> CYP20A1 mutant sequence 2

<400> 27

cttccactgt tcacaatctg g 21

<210> 28

<211> 17

<212> DNA

<213> CYP20A 1-mutant sequence 3

<400> 28

cttccactgt tcactgg 17

<210> 29

<211> 19

<212> DNA

<213> CYP20A 1-mutant sequence 4

<400> 29

cttccactgt tcacaatgg 19

<210> 30

<211> 23

<212> DNA

<213> original sequence SIRT3

<400> 30

aagcccgaca tcgtgttctt tgg 23

<210> 31

<211> 22

<212> DNA

<213> SIRT 3-mutant sequence 1

<400> 31

aagcccgaca tcgtttcttt gg 22

<210> 32

<211> 21

<212> DNA

<213> SIRT 3-mutant sequence 2

<400> 32

aagcccgaca tcgttctttg g 21

<210> 33

<211> 19

<212> DNA

<213> SIRT 3-mutant sequence 3

<400> 33

aagcccgaca gttctttgg 19

<210> 34

<211> 20

<212> DNA

<213> SIRT 3-mutant sequence 4

<400> 34

aagcccgaca tgttctttgg 20

<210> 35

<211> 23

<212> DNA

<213> original sequence ANXA7

<400> 35

ggtcagtatc cttatcctag tgg 23

<210> 36

<211> 22

<212> DNA

<213> ANXA 7-mutant sequence 1

<400> 36

ggtcagtatc cttacctagt gg 22

<210> 37

<211> 20

<212> DNA

<213> ANXA 7-mutant sequence 2

<400> 37

ggtcagtatc ctcctagtgg 20

<210> 38

<211> 18

<212> DNA

<213> ANXA 7-mutant sequence 3

<400> 38

ggtcagtatt cctagtgg 18

<210> 39

<211> 20

<212> DNA

<213> ANXA 7-mutant sequence 4

<400> 39

ggtcagtatt atcctagtgg 20

Claims

1. An episomal CRISPR/Cas9 expression vector, which is characterized in that the CRISPR/Cas9 expression vector contains an S/MAR sequence, wherein the nucleotide of the S/MAR sequence is shown as SEQ ID NO:1 is shown in the specification; the S/MAR sequence is located between the coding region sequence capable of coding for a protein and the bGH poly (A) sequence in this vector.

2. The episomal CRISPR/Cas9 expression vector according to claim 1, wherein the sequence of the coding region capable of encoding a protein is the sequence of the EGFP coding region.

3. The episomal CRISPR/Cas9 expression vector according to claim 1, wherein the sgRNA sequence of the CRISPR/Cas9 expression vector is SEQ ID NO: 2-4.

4. The episomal CRISPR/Cas9 expression vector according to claim 1, further comprising a G418 resistance gene, wherein the nucleotide sequence of said G418 resistance gene is set forth in SEQ ID NO:5, respectively.

5. The method for constructing the additional CRISPR/Cas9 expression vector as claimed in claim 1, wherein the S/MAR sequence is constructed between the coding region sequence capable of coding protein and bGH poly (A) sequence in the CRISPR/Cas9 expression vector by enzyme digestion ligation or homologous recombination.

6. The construction method according to claim 5, characterized by comprising the following specific steps:

7. The construction method of claim 6, wherein the G418 resistance gene and the px330-EGFP vector in step 1) are digested with BsmBI, the large fragment of the px330-EGFP vector backbone digested with the G418 resistance gene is recovered, and the digested product is subjected to T-cleavage ₄ DNA ligase to obtain vector skeleton px330-EGFP-G418.

8. The construction method of claim 6, wherein the S/MAR sequence and the px330-EGFP-G418 vector in step 2) are digested with Bsp1407, the backbone digestion product of the px330-EGFP-G418 vector is recovered, and the digested product is subjected to T-cleavage ₄ And performing DNA enzyme connection to obtain a vector skeleton px330-EGFP-G418-S/MAR.

9. The construction method of claim 6, wherein the sgRNA sequence and the vector backbone px330-EGFP-G418-S/MAR in step 3) are subjected to BbsI enzyme digestion respectively, then the large enzyme digestion fragment and sgRNA sequence digestion product of the vector backbone px330-EGFP-G418-S/MAR are recovered, and the large enzyme digestion fragment and the sgRNA sequence digestion product are subjected to T digestion ₄ And (3) performing DNA enzyme connection to obtain an additional CRISPR/Cas9 expression vector px330-EGFP-G418-S/MAR-sgRNA.

10. Use of the episomal CRISPR/Cas9 expression vector according to any of claims 1-4, wherein said gene knockout is a gene knockout with non-disease diagnosis and treatment as a goal.