CN107267515B - CRISPR/Cas9 targeted knockout human CNE10 gene and specific gRNA thereof - Google Patents

Abstract

The invention belongs to the technical field of molecular biology and biomedicine, and particularly relates to application of a gRNA sequence based on a CRISPR/Cas9 system and a combination thereof in knocking out a conserved non-coding sequence CNE10 of a human dwarf homologous gene (SHOX) and research in the process of regulating and controlling the occurrence of a SHOX gene-related disease by the CNE 10. According to the design principle of CRISPR/Cas9, 2 most effective targets are respectively designed on the upstream and downstream of a CNE10 genome, a sequence table of the targets is shown as SEQ ID NO.1-4, then the targets are respectively constructed on a px458 vector, and 1 guide RNA (gRNA) is obtained by respectively screening the upstream and downstream of a CNE10 genome. The 2 gRNA mediated CRISPR/Cas9 system is utilized in human osteosarcoma cells (U2 OS), the SHOX conserved non-coding sequence CNE10 can be effectively knocked out, and the system is simple and convenient to operate, high in knocking-out efficiency and suitable for cell models for researching multiple functions of genes. The gRNA related to the invention is expected to be popularized and applied in the treatment of CNE10 regulation and control SHOX gene related diseases (chondrocyte dysplasia, autism, Rett syndrome and pancreatic hypoplasia).

Description

CRISPR/Cas9 targeted knockout human CNE10 gene and specific gRNA thereof

Technical Field

The invention belongs to the technical field of molecular biology and biomedicine, and particularly relates to a CRISPR/Cas9 specific human SHOX conserved non-coding sequence CNE10 knocking out method and a gRNA combination for targeting CNE 10.

Background

With the advent of post-genomics (functional genomics), the functional identification and characterization of noncoding DNA sequences in vertebrate genomes has become a challenge and research hotspot in the post-gene era. With the progress and development of experimental techniques, these CNEs, which were regarded as garbage, are now regarded as having important biological functions. Although CNEs do not encode proteins, rRNA, tRNA, or ncRNA, and chemicals related to transcription and translation, etc., it has been found that CNEs remotely regulate gene expression in higher order structures (chromatin conformation, protein modification, etc.), RNA translation and processing, and DNA transcription levels, etc., and have also been shown to be associated with human diseases and various appearance morphologies of mammals. With the progress of research on CNEs, it is found that a large number of diseases occur due to the changes of CNEs, such as autism, Rett syndrome and pancreatic dysplasia, which are all proved that CNEs in the internal or flanking region of pathogenic genes have a regulating effect on related genes. The mutation or deletion of the internal and/or upstream and downstream PAR1 including SHOX enhancing element of the SHOX gene is also reported in both Leri-Weill dyschondroplasia, Langer acrodysplasia and idiopathic dwarf.

The inventors previously intervened in CNEs on HEK293 cells and showed no statistical difference between the untransfected group (without any intervention on the cells) and the control group (with the addition of empty plasmids without CNEs). That is, the plasmid itself has no influence on the expression level of the SHOX gene, and the influence of the plasmid on the cell and the expression of the SHOX gene is eliminated. The CNE2, CNE3, CNE5, CNE9, CNE10 and CNE11 are different from untransfected groups in statistics, namely, after the CNEs interfere cells, the expression of the SHOX gene is influenced (namely, the expression shows an enhancement or inhibition effect),

therefore, the inventor develops a gRNA of a conserved non-coding sequence CNE10 gene which has high efficiency and targeted blocking effect on SHOX gene expression, and the gRNA has extremely important effect on the research of the full play and gene function of a CRISPR/Cas9 system.

Disclosure of Invention

The invention aims to finally provide a plurality of high-efficiency gRNAs based on CRISPR/Cas9 systems, and simultaneously target human SHOX conserved non-coding sequence CNE10 and target site sequences thereof through design, construction and screening, so as to inhibit the regulation and control effect of CNE10 on SHOX gene expression, thereby controlling or treating the occurrence and development of related diseases.

In order to achieve the purpose, the invention designs a series of gRNAs based on a CRISPR/Cas9 system principle and a gRNA design principle thereof by software design prediction, and constructs a gRNA/Cas9 expression system by taking px458 as an expression vector. Through screening and series analysis and testing, 2 effective gRNAs are screened finally, and a human osteosarcoma cell (U2 OS) is utilized to prepare a human osteosarcoma cell conserved non-coding sequence CNE10 defective cell model, which has great application prospect in the field of researching diseases related to the expression of the SHOX gene remotely regulated and controlled by CNE 10.

The technical scheme of the invention is as follows:

1. designing a high-efficiency gRNA of a targeted human SHOX conserved non-coding sequence CNE10 and a target sequence thereof, and constructing a gRNA/Cas9 expression system.

2. The endogenous activity of gRNAs is analyzed and detected in a human osteosarcoma cell (U2 OS) model, 2 effective gRNAs are screened, a conservative non-coding sequence CNE10 can be successfully targeted and knocked out, and the corresponding DNA sequence is shown as any one of SEQ ID number 1 and SEQ ID NO. 4.

Drawings

FIG. 1 shows the original sequencing results of a target sequence, which are, from top to bottom, px458-CNE10-T1, px458-CNE10-T2, px458-CNE10-T3 and px458-CNE 10-T4;

FIG. 2 shows the result of agarose gel electrophoresis analysis of the PCR product digested with T7E 1;

FIG. 3 is a graph showing the results of cell cloning;

FIG. 4 is a Western-blot detection SHOX protein expression result chart.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers.

Example 1 gRNA Synthesis and vector construction targeting the human SHOX conserved non-coding sequence CNE10 Gene

1. Selection and design of gRNAs targeting the human SHOX conserved non-coding sequence CNE10 gene

The sequence of the human CNE10 gene is found in Genebank, and potential target sites are designed in the upstream and downstream regions of the human CNE10 gene exon. The gRNA is designed by evaluating a target site with higher score on a human CNE10 gene sequence through an online design tool (http:// crispr. mit. edu /) and a design principle of the gRNA, and the target site sequence is shown as SEQ ID NO. 1-4.

2. Synthesis of gRNA oligonucleotide sequence of target human SHOX conserved non-coding sequence CNE10 gene and construction of eukaryotic expression vector

The plasmid pSpCas9(BB) -2A-GFP (PX458) (AddgeneplansiidID: 48138) was digested with BbSI, subjected to 1% agarose electrophoresis at 37 ℃ for 1 hour, and the digested product was recovered (TAKARA gel recovery kit).

The enzyme digestion system is as follows:

annealing two oligonucleotides corresponding to the target site sequence to form a short double-stranded DNA with sticky ends, wherein the reaction system is as follows:

the reaction system is uniformly mixed in a 200 mu LPCR tube, then the PCR tube is treated in a water bath kettle at 37 ℃ for 30min, and then is put into 500mL of boiling water and is naturally cooled to the room temperature.

The linking system is as follows:

the double-stranded short DNA product with sticky ends is ligated into the digested pSpCas9(BB) linear fragment, the ligation product is transformed into Escherichia coli DH5 alpha competent cells (Takara code: D9057A), and the cells are spread on an LB solid plate with Ampicillin concentration of 100. mu.g/mL for overnight culture, well-grown monoclones are picked up and cultured in an LB liquid medium with 15 mM Ampicilin concentration of 100. mu.g/mL with shaking at 37 ℃ for overnight culture, plasmid colonies are extracted, and the results are shown in figure 1.

3. Preparation of endotoxin-free plasmid DNA

A. And adding 1 mu L of the constructed plasmid into 100 mu LDH5 alpha competent cells, uniformly blowing, standing for 20min in ice, then putting into a 42 ℃ water bath for 90s, quickly putting into an ice bath for 3min, adding 500 mu LLB liquid culture medium, putting into a shaking table for 180rpm and 37 ℃ for 1h, uniformly coating 100 mu L of the bacterial liquid on an LB solid culture medium with Ampicillin concentration of 100 mu g/mL, and culturing overnight at 37 ℃.

B. Taking a single colony to be cultured in LB liquid culture medium with the concentration of 3mLAmpicillin of 100 mu g/mL by shaking at the temperature of 37 ℃ at the rpm of 250 for 8 hours; taking 300 mu L of bacterial liquid, inoculating the bacterial liquid into LB liquid culture medium with 300mLAmpicillin concentration of 100 mu g/mL, and carrying out shaking culture at 250rpm and 37 ℃ for 12-16 hours;

C. collecting bacterial liquid, centrifuging at 4 deg.C and 4000rpm for 15min, discarding supernatant, collecting thallus, and extracting Plasmid according to QIAGENENDEER Plasmid MaxiKit kit instruction to obtain endotoxin-free Plasmid.

EXAMPLE 2 transfection of human osteosarcoma cells (U2 OS)

3 days before transfection, human osteosarcoma cells (U2 OS) were recovered, and the cells were cultured in a culture flask containing complete medium at 37 ℃ in a 5% CO2 incubator, and the recovered cells were subcultured one day before transfection.

Completely sucking a culture medium in a T75 bottle for culturing U2OS cells, adding 0.25% pancreatin taken out by a 2mL 4 ℃ refrigerator, enabling the pancreatin to uniformly cover the bottom of the bottle, placing the bottle in a 37 ℃ incubator for 3-5 min, taking out, shaking to find that the cells are separated from the bottom, shaking all the pancreatin, adding a U2OS complete culture medium preheated in 3mL37 ℃ water bath, blowing by using a 10mL pipette for 6-8 times without dead corners, aligning the pipette to a culture port when the pipette is difficult to blow, and blowing the culture medium by small force to cover the cells close to the bottle mouth. Then, all cells were aspirated out, placed in a 15mL centrifuge tube, 50 μ L of the mixed cells were placed in a 1.5m Leppendorf tube, 450mLU2OS complete medium, i.e., 10-fold dilution, mixed, and 10 μ L of the cells were counted in a counting plate. Recording as the first day on the day of passage, and paving 900-1000 ten thousand/T75 if transfection is carried out on the second day; if transfection is carried out on the third day, 350-400 ten thousand/T75 are paved. U20S complete medium was added per flask of T75. The cell density is observed on the day of transfection, and transfection can be carried out when the cell density is 80% -90% full.

Lipofectamine ™ 2000 (invitrogen) was transfected with the constructed transfection U2OS cells and reagents by lipofection, and the detailed procedures for transfection were as described in the transfection instructions.

After 48 hours of transfection, pancreatin is used for digesting the cells attached to the wall after transfection, the cells are collected by centrifugation, the waste liquid is sucked off, 1mL PBS is added for resuspending the cells, 500 muL of the cells are taken and placed in an original bottle for continuous culture, and the rest cells are placed in a 1.5mL centrifuge tube for DNA extraction (according to the instruction of a DNA extraction kit).

The extracted DNA is taken as a template (untransfected cell DNA is taken as a control group), a target sequence is amplified, the sequences of an amplified upstream primer and an amplified downstream primer are shown in SEQ ID NO.7-8, and a PCR reaction system is as follows:

PCR amplification procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 40s, extension at 72 ℃ for 5min after 30 cycles, and finally heat preservation at 4 ℃.

The PCR product was digested with T7 EndonuclearseI 37 ℃ water bath for 1h in the following manner:

the cleavage result of T7 Endonuclease I is shown in FIG. 2, and the result shows that the target sequence has mutation and high activity, and according to the electrophoresis result, Indel (insertion-deletion) analysis is performed, wherein px458-CNE10-T1 is (21.3%), px458-CNE10-T2 is (6.5%), px458-CNE10-T3 is (7.3%), and px458-CNE10-T4 is (14.. 8%).

According to the results of the target activity identification screening, a target point px458-CNE10-T1 with higher activity is selected at the upstream of the CNE10 gene, and a target point px458-CNE10-T4 with higher activity is selected at the downstream. Therefore, we selected px458-CNE10-T1 and px458-CNE10-T4 to transfect U2OS cells simultaneously.

According to the activity identification screening result of the designed target, the upstream of the CNE10 gene selects the target CNE10target-1 with higher activity, the downstream selects the CNE10target-4, and transfects the U2OS cell.

Cell clone culture

The CNE10target-1 and CNE10target-4 transfected cells are respectively diluted and cultured, single cell clones are cultured and used for screening and knocking out CNE10 positive cells, and the process of cell monoclone gradually increases as shown in figure 3.

Example 3 PCR product cloning sequencing detection of target site mutations

Expanding and culturing cell clone, taking partial cells, extracting cell clone genome, PCR amplifying OLIGO target site sequence, sequencing and screening (see TaKaRaMiniBEST Universal Genomic DNA Extraction KitVer.5.0 for detailed steps)

The target site sequence is PCR amplified.

The PCR product was purified using TAKARA kit and ligated into PMD18-T vector as follows:

ligation was carried out at 16 ℃ for 2 hours. And (3) taking the competent cell DH5 alpha, placing the competent cell DH5 alpha in ice to melt for 5min, adding 10 mu L of the ligation product, uniformly blowing the ligation product, and placing the ligation product in ice for 20 min. Heating at 42 deg.C for 90s, rapidly transferring into ice bath, standing for 3min, adding 500 μ L LB liquid culture medium, placing in shaking table, and heating at 37 deg.C under 180rpm for 1 h. 100. mu.L of the culture broth was applied to LB solid medium (containing 1/1000 AMP) and cultured overnight at 37 ℃.

5 single colonies were picked and placed in 3mL LB liquid medium (containing 3. mu.L AMP) at 37 ℃ for 12h at 200rpm, and 1. mu.L of the bacterial solution was used as a template for PCR identification, and all were positive. The bacterial liquid is sent for sequencing, and the CNE10 gene is successfully knocked out. The sequencing result shows that one allele of CNE10 has gene deletion/mutation of 196 bases compared with the CNE10 gene wild type control, and the corresponding sequence is shown as SEQ ID NO. 5; compared with the CNE10 gene wild type control, the other allele of the CNE10 after mutation has gene deletion/mutation of 384 bases, and the corresponding sequence is shown as SEQ ID NO. 6.

Example 4 Western-blot detection of SHOX protein expression

1. Total protein extraction

Cultured cell lysis

(1) U2OS adherent cells, medium removed, washed once with PBS, cells suspended, collected by centrifugation, washed once with PBS.

(2) Usually, 0.1 ml RIPA buffer is added to every 106 cells, and the lysate is brought into intimate 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 12,000 Xg and 4 ℃ for 5 minutes, and taking the supernatant to perform 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 12,000 Xg and 4 ℃ for 5 minutes, and taking the supernatant to perform subsequent electrophoresis, Western or immunoprecipitation.

2. Protein concentration determination (BCA protein concentration determination)

Preparation of working fluid

(1) Before measurement, a working solution is prepared by mixing BCA Reagent A and BCA Reagent B = 100: 1, for example, 30 ml of the working solution is prepared, 0.3 ml of BCA Reagent B is added to 30 ml of BCA Reagent A, and the mixed working solution is sufficiently shaken and stored at 4 ℃ for three days.

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

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

Example) standard procedure [ 1ml reaction system ] when the number of samples tested is 12, parallel samples (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). times.2 + 1 ]. times.0.5 ml ═ 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 0.2 mg/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 according to the following table, 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 562 nm 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 562 nm 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) By detecting the expression of the SHOX protein in the transfected U2OS through Western-blot, the expression of the SHOX protein in the CNE10 gene knockout group is obviously reduced compared with that in a control group without gene knockout, and is only 62.1 percent of the expression level of the SHOX protein in normal human osteosarcoma cells.

SEQUENCE LISTING

<110> Chongqing medical university affiliated children hospital

<120> CRISPR/Cas9 targeted knockout human CNE10 gene and specificity gRNA thereof

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Claims (3)

1. A gRNA combination characterized by: the gRNA combination is used for CRISPR-Cas9 specific knockout of a gRNA combination of a conserved non-coding sequence CNE10 of a SHOX gene of human osteosarcoma cell U2OS, and the sequence of the gRNA combination is shown as SEQ ID No.1 and SEQ ID No. 4.

2. A cell model, comprising: the cell model is a defect cell model of human osteosarcoma cell conserved non-coding sequence CNE10 prepared by using human osteosarcoma cell U2OS and named as U2OS-CNE 10; the mutant CNE10 sequence in the cell model is obtained by using the gRNA combination knockout in claim 1, and the mutant CNE10 sequence is shown in SEQ ID NO.5 and 6.

3. Use of a gRNA combination according to claim 1 in the preparation of an agent for the treatment of a disease associated with CNE10 regulation of SHOX gene, characterized in that: the diseases include chondrocyte dysplasia, autism, Rett syndrome and pancreatic hypoplasia.

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