CN108913691B

CN108913691B - Card3 gene knockout in epidermal stem cells by using CRISPR-Cas system

Info

Publication number: CN108913691B
Application number: CN201810780112.1A
Authority: CN
Inventors: 杨骏; 朱成光
Original assignee: Shandong Huayu Biotechnology Co Ltd
Current assignee: Shandong Huayu Biotechnology Co., Ltd
Priority date: 2018-07-16
Filing date: 2018-07-16
Publication date: 2020-09-01
Anticipated expiration: 2038-07-16
Also published as: CN108913691A

Abstract

The invention provides a method for carrying out gene editing of Card3 on epidermal stem cells by using a CRISPR-cas system, and particularly relates to establishment of an epidermal stem cell line with a knocked-out Card3 gene. Two specific gRNAs are constructed and obtained, and the gene editing efficiency of CRISPR/Cas9 in epidermal stem cells to Card3 can be obviously improved. The epidermal stem cell Card3 knockout plasmid provided by the invention has better genetic stability and higher targeting efficiency.

Description

Card3 gene knockout in epidermal stem cells by using CRISPR-Cas system

Technical Field

The invention provides CARD3 gene editing for epidermal stem cells by using a CRISPR-cas system, and particularly relates to establishment of an epidermal stem cell line with a CARD3 gene knockout structure.

Background

Epidermal stem cells (epistem cells, EpiSCS) are stem cells with the potential for self-proliferation and multidirectional differentiation, the normal proliferation and differentiation of which are essential requirements for maintaining the structural and functional integrity of the skin and its appendages (sweat gland hair, sebaceous glands). Under physiological conditions, the epidermal stem cell is differentiated into a stem cell and a transient amplifying cell (TA cell) by an asymmetric division mode, and the TA cell is differentiated into a Post-mitotic cell (Post-mitotic cell) and a terminal differentiated cell (terminal-differentiated cell) after multiple divisions so as to meet the requirement of continuous renewal of the epidermal cell. Studies have shown that epidermal stem cells not only can be subcultured for long periods in vitro and retain their proliferative differentiation potential (Dunnwald et al, Exp dermaltol, 2001, 10: 45-54.Papini et al stem cells, 2003, 21: 481-: 20: 21-31]. Therefore, the purified epidermal stem cells can be obtained, which not only can provide seed cells for constructing artificial skin with physiological functions, but also can be used for gene therapy and the production of transgenic animals.

Human pluripotent stem cells (hPSCs) and genome editing technology are combined to establish a cell model, and a unique experimental platform is provided for disease research. By utilizing the platform system, researchers can research the influence of specific gene mutation and even chromosome structure variation on various cell types and tissue and organ functions of human beings and detailed molecular mechanisms thereof, and can establish 'personalized' disease models carrying different genetic mutations for large-scale drug screening. The establishment of the model system benefits from the rapid development of a genome editing technology, in particular a CRISPR/Cas9(Clustered regulated short linked templates/CRISPR-associated proteins9, CRISPR/Cas9) technology.

Card3(Caspase activator 1n and receptor domain 3), also known as RIP2, receptor interacting protein 2(receptor _ interacting protein 2), is a member of the RIP family, is distributed in many tissues, is a silk/threonine protein kinase, a mediator domain between the kinase domain and the Card domain, and was originally discovered in 1998 when screening RIP and Card homologues by 3 independent groups, and Card3 is distributed in the cytoplasm and is widely expressed in tissues, with mRNA highly expressed in tissues such as spleen, peripheral blood leukocytes, placenta, testis, and heart. Card3 plays an important role in innate immunity, adaptive immunity, and inflammatory responses, and is involved in a variety of disease processes. Many studies have found that when infected with bacteria and viruses, Card3 mediates the activation of NF- κ beta and causes an inflammatory response, and particularly in the N0D/RIP2 signaling pathway, Card3 as its downstream factor causes an appropriate immune response in the host. A significant feature of Card3 is that its expression is regulated at the transcriptional level in a cell-specific, time-dependent manner. Increased expression of Card3 is in response to cell-specific stimuli, including bacterial peptidoglycans and lipopolysaccharides.

CN103877576A discloses the function and application of Caspase activation and recruitment binding domain 3(Card3) gene in coronary atherosclerotic heart disease. According to the invention, a Card3 gene knockout mouse and a heart-specific Card3 transgenic mouse are taken as objects, and a myocardial infarction model caused by blocking the anterior descending of the left coronary artery of the heart of the mouse is researched, and the result shows that compared with a WT mouse, the heart infarction proportion, the myocardial hypertrophy and the fibrosis degree of the Card3 gene knockout mouse are obviously inhibited, and the cardiac function is obviously improved; the phenotype of the mouse transgenic with the Card3 gene is opposite to that of the mouse knockout with the Card3 gene. The gene Card3 is shown to be capable of promoting and aggravating the occurrence and development of coronary atherosclerotic heart disease, Card3 can be used as a drug target for screening drugs for treating coronary atherosclerotic heart disease, and an inhibitor of Card3 can be used for preparing drugs for treating coronary atherosclerotic heart disease.

Based on the pluripotency of the epidermal stem cell, in order to research the functions of the epidermal stem cell with the CARD3 gene knocked out in the aspect of heart disease treatment, the establishment of the epidermal stem cell line with the CARD3 gene knocked out becomes particularly important.

Disclosure of Invention

The invention aims to provide an epidermal stem cell with a CARD3 gene knocked out, which effectively overcomes the technical defect that the prior art uses siRNA for interference and can not stably inherit.

In order to achieve the above object, the present invention provides a target of CRISPR-cas system, and according to the gene sequence of CARD3, specific sgrnas are designed as follows:

CARD3-sgRNA1：5’to 3’agtagcaaatctgcaccaga

CARD3-sgRNA2：5’to 3’ataatgtatagtgtgtcaca。

to achieve the above object, the present invention provides a method for improving CRISPR-cas system to knock out CARD3 gene in epidermal stem cells, comprising introducing into host cells a synergistic protein, wherein the synergistic protein ESCS-higher is a protein encoded by the nucleotide sequence shown in SEQ ID NO. 1.

Further, the potentiating protein comprises a) or b):

a) 1, and a polynucleotide sequence of a protein coded by the nucleotide sequence shown in SEQ ID NO;

b) the amino acid sequence shown in SEQ ID NO. 2.

Still further, there is provided a system for gene editing using CRISPR/Cas9 in epidermal stem cells, characterized in that the system comprises: (1) for expressing SEQ ID NO:1 the plasmid of ESCS-higher; (2) a PX 330-expressing plasmid (which can express sgRNA and cas9) into which sgRNA has been inserted.

Further, the sgRNA and cas9 expression vectors may also be other expression vectors commonly used in the art.

The invention provides a method for editing CARD3 by using CRISPR/Cas9 gene in epidermal stem cells, which has the following advantages:

the invention provides 2 specific gRNAs which can specifically knock out the CARD3 gene in an epidermal stem cell by designing and optimizing the gRNAs for nearly one hundred times, and the gRNAs have strong knock-out effects.

Drawings

FIG. 1 is a graph of a result of detecting CARD3 protein expression by Western-blot; 4 is epidermal stem cell blank control; 3 represents the effect of gRNA1 into which no potentiating protein was introduced; 1 is the effect of gRNA1 introduced with a potentiating protein; 2 shows the effect of gRNA2 introduced with a potentiated protein.

The technical solution of the present invention is further described in detail by the following examples.

Detailed Description

The technical scheme of the method for improving the genome editing efficiency of the invention is further illustrated by the specific examples.

Example 1 construction of CRISPR expression vectors

Design of gRNA

According to the gene sequence of a target gene, the specific form of sgRNA obtained by the applicant through early optimization design and specific screening from dozens of grnas is as follows:

CARD3-sgRNA1：5’to 3’agtagcaaatctgcaccaga

CARD3-sgRNA2：5’to 3’ataatgtatagtgtgtcaca

according to the above gRNA, a forward oligonucleotide sequence was obtained by adding CACC to the 5 'end of the gRNA, a reverse oligonucleotide sequence was obtained by adding AAAC to the 5' end of the complementary strand, and the forward and reverse oligonucleotide sequences were synthesized, respectively, and then the synthesized sequences were denatured and annealed to obtain a double-stranded DNA fragment having a BbsI cohesive end, as follows: forward direction: 5' -CACCNNNNNNNNNNNNNNNNNNNN reverse: NNNNNNNNNNNNNNNNNNNNCAAA-5', the denaturation and annealing system is as follows: mu.l forward oligonucleotide strand (50. mu.M) 2. mu.l reverse oligonucleotide strand (50. mu.M) 46. mu. l l. mu.NEB buffer was run in a PCR instrument following the following procedure: 4min at 90 ℃; 72 ℃ for 10 min; 22min at 37 ℃; 25 ℃ for 25 min.

The annealed double-stranded oligonucleotide chain contains a cohesive end of BbsI, and is directly connected with a pX330-U6-Chimeric _ BB-CBh-hSpCas9 (hereinafter referred to as PX330) vector cut by BbsI, so that a PX330-gRNA-Cas9 recombinant plasmid can be obtained.

Enzyme digestion system: 39.3. mu.l of water, 5. mu.l of 10. mu.l of FD buffer, 2. mu.l of BbsI, and 2. mu.l (2. mu.g) of PX 3303.7. mu.l (2. mu.g) digested plasmid in 37 ℃ water bath for 2h were recovered directly by using a gel recovery kit.

A connection system: annealing product 0.5 μ l, linearized PX330 plasmid 2 μ 1, 5. mu.l ligation buffer2 μ l, T4DNA Ligase (3 units/. mu.1), 1 μ l, water 4.5 μ l, water bath 2h at 16 ℃ transforming the ligation product obtained in the above steps into JM109 competent cells, coating on LB plate of Amp +, picking positive clone inoculation, shaking table bacteria at 37 ℃ overnight, extracting plasmid with plasmid extraction kit and sequencing to obtain PX330-gRNA plasmid.

Example 2 cloning of the synergistic protein ESCS-higher and construction of the vector

Cloning the synergistic protein ESCS-higher gene, and obtaining the amino acid sequence shown in SEQ ID NO:1, the gene sequence is used as a template, the sequences of upstream and downstream primers are 5'-atgatatactttattagaat-3' and 5'-tcaagggatttccatttctc-3' respectively, and the primers and the whole genome are synthesized by Shanghai Biotech Co., Ltd. And (2) amplifying the ESCS-higher gene target gene fragment by PCR reaction, wherein the amplification reaction system comprises 95 ℃, 40s, 57 ℃, 1min, 72 ℃, 10min and 35 cycles, PCR products are sequenced by Shanghai's Productivity Co., Ltd, and the PCR products are combined with the sequences shown in SEQ ID NO:1 are perfectly matched. And then, connecting the target gene amplified by the PCR to an empty vector lentiviral vector pHIV-CS-CDF-CG-PRE, and identifying the recombinant lentiviral vector by methods of PCR amplification, enzyme digestion, sequencing and the like. The combination proves that the recombinant lentiviral vector is successfully constructed. The recombinant lentiviral vector plasmids were then co-infected with helper plasmids (ESCs) (isolated and cultured according to the method of claim 1 in CN 1253558C) and packaged by recombination into epidermal stem cells expressing the ESCS-higher gene. Stably transfected stem cells were used for subsequent gene editing applications as identified by PCR screening.

Example 3 analysis of the use of CRISPR/Cas9 in epidermal Stem cells

The sgRNA expression plasmid prepared in example 1, the well-known Cas9 expression plasmid, was co-transfected into epidermal stem cells. Lipofectamine is prepared by transfecting the constructed epidermal stem cell with liposome transfection method and using Lipofectamine as reagent^TM2000 (invitrogen) and the detailed steps of transfection are described in the instructions for transfection. Stem cells not transfected with the enhanced gene of example 2 were used as positive controls.

Example 4 detection of CARD3 protein expression situation by Western-blot

1. Total protein extraction

Cultured cell lysis

(1) And (3) removing the adherent cells of the epidermal stem cells, removing the culture solution, washing once with PBS, suspending the cells, centrifugally collecting, and washing once with PBS.

(2) Usually, 0.1ml RIPA buffer is added to every 106 cells, and the lysate is brought into contact with the cells.

(3) The cells were fully lysed by gentle blowing with a pipette tip after a few minutes on ice, and the lysate was then poured onto one side or corner of a petri dish by gentle tilting, then transferred to a 1.5ml centrifuge tube and shaken vigorously for 30 seconds.

(4) Centrifuging at 4 deg.C for 5min at 12,000 Xg, and collecting supernatant for subsequent electrophoresis, Western or immunoprecipitation.

Tissue mass lysis

(1) The tissue is sheared into fine fragments. 1ml of RIPA lysate was added per 100 mg of tissue. Homogenize manually 20 times up and down with a glass homogenizer.

(2) The homogenate was transferred to a 1.5ml centrifuge tube.

(3) Centrifuging at 4 deg.C for 5min at 12,000 Xg, and collecting supernatant for subsequent electrophoresis, Western or immunoprecipitation.

2. Protein concentration determination (BCA protein concentration determination)

Preparation of working fluid

(1) Before the measurement, the mixture was mixed at a ratio of BCA Reagent A to BCA Reagent B of 100:1 to prepare a working solution, and for example, when 30ml of the working solution was prepared, 0.3ml of BCA Reagent B was added to 30ml of BCA Reagent A, and the thus prepared working solution was sufficiently shaken and mixed, and then stored at 4 ℃ for three days.

(2) The required working fluid amount is calculated as follows:

total volume (ml) of working solution required to be [ (8 parts or 7 parts of BSA standard solution + number of samples to be detected) × parallel number of samples (n) +1] × 1 sample

Example) standard procedure [ 1ml reaction system ] when the number of samples tested is 12, replicates (n ═ 2):

[(8+12)×2+1]×1ml＝41ml

example) standard procedure [ 200 μ l reaction system ], number of samples tested 20, replicates (n ═ 2):

[(8+20)×2+1]×0.2ml＝11.4ml

example) procedure for low concentration protein sample determination [ 1ml reaction system ], number of samples tested 12, parallel sample (n ═ 2): [ (7+12) × 2+1] × 0.5ml ═ 19.5ml

3. Standard operation procedure of low concentration protein sample (quantitative range: 0-200 mug/ml)

[ 0.2ml reaction System, assay Using microwell plates ]

1) And (3) preparing a BSA standard solution.

(1) Preparation of 0.2mg/ml BSA standard solution: mu.l BSA Standard Solution (2mg/ml) was added to 1,080. mu.l of the dilution and mixed well.

(2) The BSA standard solution was diluted at different concentrations, and deionized water, 0.9% NaCl, or PBS was used for dilution of the BSA standard solution and the test sample.

2) Preparation of BSA Standard Curve

(1) 100. mu.l of the diluted BSA standard solutions were added to the plate, and 2 replicates were taken at each concentration.

(2) After 100ul of working solution was added, the mixture was immediately mixed.

(3) After reacting in a 37 ℃ water bath for 60 minutes, the mixture was cooled to room temperature.

(4) The absorbance value at 562nm was measured using a spectrophotometer. For the measurement, a 1mL cuvette was used and the water was used for zero calibration. All samples were tested as soon as possible within 20 minutes.

(5) The absorbance value of the BSA standard solution at each concentration was subtracted by the average value of the Blank values, and a standard curve of the BSA standard solution was plotted.

3) Determination of test sample

When the sample is detected, the sample is recommended to be detected simultaneously with the BSA standard solution.

(1) 100 mul of each sample was added to the microplate and 2 replicates of each sample were tested.

(if necessary, measurement can be made after diluting the test sample by the same dilution method as that for the BSA standard solution)

(2) After adding 100. mu.l of the working solution, mix well immediately.

(3) After reacting in a water bath at 37 ℃ for 60 minutes, the reaction mixture was cooled to room temperature.

(4) The microplate reader wavelength was set at 562nm for the determination. Zero calibration with water. All samples were tested as soon as possible within 20 minutes.

(5) The absorbance value of each sample solution was subtracted by the average value of Blank values, and the protein concentration of the test sample was calculated from the standard curve.

SDS-PAGE electrophoresis

(1) The glass plates are aligned and then placed into a clamp for clamping. The card is then vertically mounted on a rack ready for potting.

(2) Preparing 10% separation gel, adding TEMED, immediately shaking up, and filling gel.

(3) When there is a line of refraction between the water and the gel, the gel is said to have solidified. Waiting for 3min for the glue to solidify sufficiently, pouring off the water on the upper layer of the glue, and sucking the water with absorbent paper.

(4) 4 percent of concentrated glue is prepared, and the concentrated glue is immediately shaken up after TEMED is added, so that glue can be filled. The remaining space was filled with the gel concentrate and a comb was then inserted into the gel concentrate.

(5) The concentrated gel was washed with water and placed in an electrophoresis tank. (the small glass plate faces inwards, the large glass plate faces outwards, if only one piece of glue is run, a plastic plate is padded on the other side of the groove, and the side with the character faces outwards.)

(6) The sample was removed and mixed with 5 x SDS loading buffer as 4: mixing at a ratio of 1, mixing, and decocting in boiling water for 5min to denature protein.

(7) Adding sufficient electrophoresis solution, and loading the same amount of protein.

(8) And (4) electrophoresis, wherein after 80V runs through the concentrated gel, the voltage is converted to 120V, and when bromophenol blue runs to the bottom of the gel plate, the bromophenol blue just does not run out.

(9) Opening the clamp to keep the black side horizontal, and sequentially filling a sponge pad, filter paper, glue, a PVDF membrane (activated by methanol), the filter paper and the sponge pad on the clamp; and simultaneously, the electrophoretic solution is changed into transfer solution.

(10) The current was regulated to a constant current of 200mA for about 1 hour of transfer.

(11) The membrane was removed and front and back labeled, washed in TBST for 1 minute, and then blocked with blocking solution.

(12) Diluting the corresponding primary antibody to a certain concentration (1:500) by using a confining liquid, wherein the final dilution concentration of the internal reference primary antibody is 1:3000, then incubated for 1.5 hours or overnight at 4 ℃.

(13) The washing was performed 3 times for 5 minutes each with TBST.

(14) The secondary antibody was diluted to a certain concentration (1:3000) with blocking solution and then incubated for 1.5 hours.

(15) Wash 4 times 5 minutes each with TBST.

5. Chemiluminescence, development, and fixation

(1) The reagents A and B were mixed in equal volumes in a test tube and then applied to the front of the PVDF membrane and incubated for approximately 2 minutes.

(2) And (4) entering a dark room, covering a layer of preservative film on the PVDF film, and wiping off the redundant luminous agent. The film is pressed on the preservative film, and different exposure time is selected according to the intensity of the light emission.

(3) And (3) putting the film into a developing solution, immediately putting the film into a fixing solution after a strip appears, washing the film with running water, and drying the film.

(4) The film was scanned and the grey values of the bands of interest were analyzed using the UVP gel image processing system labworks4.6 software.

(5) Detecting the expression of CARD3 protein in the transfected epidermal stem cells by Western-blot, wherein the result is shown in figure 1, and the expression of the protein is not influenced by the blank control of the epidermal stem cells; the gRNA1 without the introduced synergistic protein can knock out a target gene, protein expression can be well inhibited, and the inhibition rate reaches 82.6%; the gRNA1 introduced with the synergistic protein has the obvious effect of improving the gene knockout effect and the protein inhibition rate reaches 99.2 percent; the protein expression inhibition rate of gRNA2 introduced with the synergistic protein reaches 99.0%, and the effect is also extremely remarkable. Has excellent application prospect and application value.

Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

<110> Luoyang Xuan Zhi Biotech Co., Ltd

<120> performing Card3 gene knockout in epidermal stem cells by using CRISPR-Cas system

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Claims

1. A CRISPR-CAS system for epidermal stem cell Card3 gene editing, characterized in that: the system comprises the following components: (1) for expressing SEQ ID NO:1, the plasmid of ESCS-higher; (2) expresses SEQ ID NO: 3 or 4, sgRNA shown and PX330 plasmid of Cas 9.

2. The system of claim 1, wherein: (1) the plasmid (2) is introduced into a gene-editing cell in advance, and after a positive cell is obtained by screening, the plasmid (2) is transferred.

3. Use of the system of claim 1 in the preparation of a reagent for epidermal stem cell Card3 gene editing.