CN104651398A - Method for knocking out microRNA gene family by utilizing CRISPR-Cas9 specificity - Google Patents

Abstract

The invention discloses a method for specifically knocking out a microRNA gene family by using CRISPR-Cas9, mainly using the CRISPR/Cas9 system to knock out a target gene. This is the first time that the micrRNA family has been specifically knocked out using the CRISPR/Cas9 system. Utilizing this new method can overcome the drawbacks of traditional transgenics that can only knock out one gene at a time, and accelerate the establishment of model organisms to 3 weeks; at the same time, the construction steps are simple, and expensive molecular reagents are also reduced; the invention constructs genetically modified The cycle of mice is greatly shortened to 4 months; multiple genes can be knocked out at the same time; capital investment is significantly reduced, and F1 generation mice can be obtained with only 50,000 yuan; the efficiency of gene modification can reach 90% The above reduces the unreliability of traditional technology; the operation technology is simple, and there is no need to go through a series of steps such as complex targeting vector construction, ES cell screening, and chimera mouse breeding.

Description

A method for specifically knocking out microRNA gene families using CRISPR-Cas9

(1) Technical field

The present invention relates to a method for specifically knocking out microRNA genes by CRISPR-Cas9 and the design of specific targeting sgRNA.

(2) Background technology

In recent years, my country's pharmaceutical industry has developed rapidly, but at the same time there are many problems, such as weak research and development foundation, flood of imitation of foreign patents, lack of independent intellectual property rights, etc., which seriously restrict the development of my country's pharmaceutical industry. Therefore, it is urgent to enhance my country's drug research and development capabilities. The drug research and development process is the process of disease pathogenic mechanism and drug target screening. Most of the screening process relies on animal disease models of various human diseases, especially mouse models that are closely related to humans (including transgenic and gene knockout) To analyze the pathogenesis of diseases and the development of new drugs. It is determined that the characteristics of mice are similar to those of humans, such as organ development, biochemical and physiological metabolism, etc., and also depends on the high reproductive rate of mice and the ease of obtaining materials, which greatly facilitates the research work. At present, my country is still late in the establishment of disease mouse models, especially in terms of genetically modified mice such as transgenic/gene knockout, only Shanghai Southern Model Organism Center and Nanjing University Model Organism Institute have limited production capacity, most of them Researchers still rely on imports, which are very expensive. Each genetically modified mouse needs about 200,000 yuan, and the cycle is long, which greatly restricts the development of my country's pharmaceutical industry. Therefore, in this context, how to establish and develop a simple, efficient, fast and cheap method for preparing genetically modified mouse models is of great significance to the development strategy of my country's pharmaceutical industry.

In the process of animal and plant development, MicroRNA, as a class of important regulatory factors, participates in the regulation of gene expression. miRNA can negatively regulate the expression of a certain gene by binding to the 3' untranslated region (UTR) of specific mRNA, leading to mRNA degradation or inhibition of translation process using mRNA as a template. The study of miRNA function is of great significance for further revealing the occurrence and development of diseases.

The method of studying microRNA is mainly to study its function by knocking out microRNA so that its function is lost. However, most microRNAs regulate gene expression in the form of microRNA families; knocking out a microRNA alone cannot completely change the function of the target gene. However, using the traditional method to construct a microRNA family knockout animal model is complicated and labor-intensive; the use of artificially synthesized microRNA antagonists to transfect cells or intravenously inject them into animals is obviously insufficient: the cost is high and repeated injections are required, among which , the more prominent is the short duration of action and can not be passed down. Experiments have shown that after cells are transfected with miRNA inhibitors, they can act for up to 7 days, which obviously cannot meet the requirements of some disease research. The inability to be passed on to the next generation restricts some studies on embryonic development. Therefore, a new genome modification technology is needed to simultaneously knock out multiple microRNAs.

CRISPR/Cas9 technology is an emerging gene modification technology, which was discovered in the long-term evolution of bacteria and archaea as an adaptive immune defense, which can be used to fight against invading viruses and foreign DNA. The principle is that crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex, which guides the nuclease Cas9 protein to cut at the sequence target site paired with crRNA double-stranded DNA. Using this system, not only can the target gene be modified efficiently, but also multiple target genes can be precisely modified at one time.

At present, the CRISPR/Cas9 system has great advantages in genome editing, and can delete target genes in a targeted manner. The present invention utilizes the technology to knock out miR-382 and miR-154 genes of the miR-382 family, and the technology has high knockout efficiency and simple operation steps. The use of this technology for genetic modification has the unique advantages of high controllability and safety, and reduces the risk of genetically modified organisms.

(3) Contents of the invention

The purpose of the present invention is to provide a method for efficient site-specific knockout of microRNA family genes, mainly using the CRISPR/Cas9 system to knock out the target gene. This is the first time that the micrRNA family has been specifically knocked out using the CRISPR/Cas9 system. Using this new method can overcome the disadvantage of traditional transgenics that can only knock out one gene at a time, and accelerate the establishment of model organisms to 3 weeks; at the same time, the construction steps are simple and expensive molecular reagents are reduced.

The technical scheme adopted in the present invention is:

The present invention provides a method for using CRISPR-Cas9 to specifically knock out microRNA genes, the method comprising: (1) using the px330 plasmid as a template, performing PCR under the action of primer Cas9-R and primer Cas9-F containing a T7 promoter Reaction, the PCR reaction product is connected with the pGME-T vector to obtain the plasmid pGEM-Cas9, and then the plasmid pGEM-Cas9 is recovered through linearization, in vitro transcription and RNA purification kit to obtain Cas9 mRNA; (2) using the px330 plasmid as a template, Carry out PCR reaction under the effect of primer sgRNA-R and the primer sgRNA-F that contains T7 promoter, then PCR amplified product is connected with vector pGEM-T, obtains vector pGEM-T7-sgRNA; (3) SEQ ID NO The miR-154sgRNA shown in .3 and the miR-382sgRNA shown in SEQ ID NO.4 were connected with the carrier pGEM-T7-sgRNA respectively to obtain the plasmid T7-sgRNA-miR-382 and the plasmid T7-sgRNA-miR-154 respectively. Linearization, in vitro transcription and recovery of RNA purification kits to obtain T7-sgRNA-miR-382mRNA and T7-sgRNA-miR-154mRNA; (4) Cas9mRNA, T7-sgRNA-miR-382mRNA and T7-sgRNA-miR-154mRNA Mixed into a mixture and injected into fertilized eggs of mammals to achieve the purpose of targeted knockout of microRNA genes;

Cas9-F: 5'-TTAATACGACTCACTATAGGATGGACTATAAGGACCACGAC-3';

Cas9-R: 5'-GCGAGCTCTAGGAATTCTTAC-3';

sgRNA-F: 5'-TTAATACGACTCACTATAGGGTGGAAAGGACGAAACACCGGGTCTTCGAGAAGACCT-3';

sgRNA-R: 5'-AAAAGCACCGACTCGGTGCC-3'.

Further, the concentrations of Cas9mRNA, T7-sgRNA-miR-382mRNA and T7-sgRNA-miR-154mRNA in each 20μl mixture were 50ng/μl, 100ng/μl and 10ng/μl, respectively.

Further, the fertilized eggs of mammals are fertilized eggs of mice.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

1) The cycle of constructing genetically modified mice is greatly shortened to 4 months;

2) Simultaneous knockout of multiple genes can be constructed at one time;

3) The capital investment is significantly reduced, and F1 generation mice can be obtained with only 50,000 yuan;

4) The gene modification efficiency can reach more than 90%, reducing the unreliability of traditional techniques;

5) The operation technique is simple, and there is no need to go through a series of steps such as complex targeting vector construction, ES cell screening, and chimera mouse breeding.

Therefore, the preparation of genetically modified mice by CRISPR/Cas9 technology has great advantages. It is simple, fast and affordable, and mouse models can be prepared with limited funds.

(4) Description of drawings

Figure 1 is a model diagram of knocking out miR-382 family genes by CRISPR/cas9.

Fig. 2 is the electrophoresis diagram of the PCR amplification product of the Cas9 expression vector, the swimming lane M is the standard molecule, and the swimming lane cas9 is the PCR amplification product of the Cas9 expression vector.

Figure 3 is the electrophoresis image of Cas9mRNA in vitro transcription products, swimming lane M is the standard molecule, and swimming lane cas9mRNA is the in vitro transcript of pGEM-Cas9 vector.

Figure 4 is the electrophoresis diagram of the PCR product of the T7-sgRNA expression vector, the lane M is the standard molecule, and the lane pGEM-sgRNA is the PCR amplification product of the sgRNA.

Figure 5 is the electrophoresis image of miR-382 family sgRNA in vitro transcription, lane M is the standard molecule, lane miR-154 is the electrophoresis image of T7-sgRNA-miR-154 in vitro transcription product, and lane miR-382 is the T7-sgRNA-miR-382 in vitro Electropherogram of transcripts.

Figure 6 is the miR-382 family deletion sequence.

Figure 7 is a picture of the heart after miR-382 knockout.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The pGEM-T vector was purchased from Promega, catalog number A3600. LA Taq was purchased from TAKARA, catalog number RR02MA.

Example 1

1. Construct the Cas9 expression vector in vitro, named pGEM-Cas9, the sequence is as SEQ ID NO.1, and the time is 3 days.

(1) Primer design

Using the px330 (Addgene:42330) sequence as a template, the primer Cas9-F (the underlined part is the T7 promoter sequence) and the primer Cas9-R were designed.

Cas9-F:

5'- ATGGACTATAAGGACCACGAC-3';

Cas9-R: 5'-GCGAGCTCTAGGAATTCTTAC-3'.

(2) PCR amplification

Using px330 as a template, PCR reaction was carried out under the action of primer Cas9-F and primer Cas9-R, and the amplified product was analyzed by gel electrophoresis, and the target fragment size was 5309bp, as shown in Figure 2.

The PCR reaction system is:

The PCR reaction conditions are:

(3) Construction of pGEM-Cas9 expression vector

Take 1 μl of the PCR product of step (2) and connect it to the pGEM-T vector, and place it at room temperature (25°C) for 1 hour. The connection system is as follows:

Take 2 μl of the connection solution to transform 50 μl Escherichia coli DH5α competent cells, ice bath for 30 minutes, heat shock at 42°C for 60 seconds, add 500 μl LB liquid medium after ice bath for 2 minutes, place on a shaker at 37°C and 200 rpm for 1 hour, and take 200 μl of culture solution for coating Cultivate overnight at 37°C on LB plates containing X-gal at a final concentration of 50 mg/ml+ IPTG at a final concentration of 24 mg/ml ⁺ Amp at a final concentration of 100 mg/ml. Pick the white spot, inoculate it in 5ml of LB liquid medium with a final concentration of 100mg/ml Amp ⁺ , shake it overnight at 37°C and 200rpm, extract the plasmid, and the sequencing results show that the pGEM-Cas9 expression vector was successfully constructed.

(4) Linearized pGEM-Cas9 vector

The pGEM-Cas9 expression vector prepared in step (3) was linearized overnight at 37°C.

The reaction system is as follows:

(5) In vitro transcription of Cas9

Take 1 μg of the linearized pGEM-Cas9 vector and use the in vitro transcription kit (ambionAM1340) for in vitro transcription, and react at 37°C for 3 hours to obtain the in vitro transcript of the pGEM-Cas9 vector. The specific transcription system is as follows:

(6) Use the RNA purification kit (ambion AM1908) to purify in vitro transcripts, the specific purification system is as follows:

The purified solution was passed through the adsorption column of the RNA purification kit, the effluent was centrifuged at 12000rpm for 30s, the precipitate was washed twice with 70% alcohol, and finally eluted with 80 μl RNase-free water, and the collected solution was analyzed by gel electrophoresis, as shown in Figure 3, the results showed that Cas9 mRNA.

2. Construction and preparation of the vector for transcribing the sgRNA backbone coding sequence in vitro, named pGEM-sgRNA, and the sequence is as SEQ ID NO.2.

The sgRNA backbone sequence with T7 promoter (ie T7-sgRNA nucleic acid fragment) was artificially synthesized, and the artificially synthesized T7-sgRNA nucleic acid fragment was cloned into the pGEM-T vector. details as follows:

(1) Design primers

sgRNA-F (T7 promoter is underlined):

5'- GTGGAAAGGACGAAACACCGGGTCTTCGAGAAGACCT-3'

sgRNA-R: 5'-AAAAGCACCGACTCGGTGCC-3'

(2) PCR amplification

Using px330 as a template, PCR reaction was carried out under the action of primer sgRNA-F and primer sgRNA-R containing T7 promoter, the PCR amplification product was analyzed by gel electrophoresis, and the target fragment size was 120bp, as shown in Figure 4.

The PCR reaction system is:

The PCR reaction conditions are:

(3) Take 1 μl of the PCR amplification product of step (2) and connect it to pGEM-T, and place it at room temperature for 1 hour.

Take 2 μl of the connection solution to transform 50 μl Escherichia coli DH5α competent cells, ice bath for 30 minutes, heat shock at 42°C for 60 seconds, add 500 μl LB liquid medium after ice bath for 2 minutes, place on a shaker at 37°C and 200 rpm for 1 hour, and take 200 μl of culture solution for coating Cultivate overnight at 37°C on LB plates containing X-gal at a final concentration of 50 mg/ml+ IPTG at a final concentration of 24 mg/ml ⁺ Amp at a final concentration of 100 mg/ml. Pick the white spot, inoculate it in 5ml of LB liquid medium with a final concentration of 100mg/ml Amp ⁺ , shake it overnight at 37°C and 200rpm, extract the plasmid, and the sequencing results show that pGEM-T7-sgRNA was successfully constructed and is ready for use. There are two BbsI restriction sites between T7 and the sgRNA sequence, and the recognition sequence for a certain gene can be inserted between the two restriction sites.

Example 2

1. Design sgRNA for mmu-miR-154 and mmu-miR-382 genes, add a BbsI restriction site (underlined) at the 5' end of the primer, and obtain miR-154 sgRNA (sequence such as SEQ ID NO.3) through denaturation and annealing reactions ) and miR-382sgRNA (sequence such as SEQ ID NO.4), the time is 2 days.

sgRNA-miR-382-F:5'- TACTTGTGACGAATCATTCA-3'

sgRNA-miR-382-R:5'- TGAATGATTCGTCACAAGTAC-3'

sgRNA-miR-154-F:5'- TATTCGTGACGAATCATACA-3'

sgRNA-miR154-R:5'- TGTATGATTCGTCACGAATAC-3'

Denaturation, annealing reaction system is as follows:

The reaction system in the PCR instrument is as follows: 37°C for 30min; 95°C for 5min; 95-25°C, 5°C/min, and the product is miR-382sgRNA.

Denaturation, annealing reaction system is as follows:

The reaction system in the PCR instrument was as follows: 37°C, 30min; 95°C, 5min; 95-25°C, 5°C/min, and the product was miR-154sgRNA.

The denatured and annealed miR-382sgRNA and miR-154sgRNA were respectively connected to the pGEM-sgRNA carrier, and reacted at room temperature for 12 hours to obtain plasmid T7-sgRNA-miR-382 and plasmid T7-sgRNA-miR-154 respectively. The reaction system was as follows:

2. Linearize the plasmids T7-sgRNA-miR-382 and T7-sgRNA-miR-154 respectively, and the reaction time is 3 hours.

The reaction system is as follows:

The DNA was then purified with phenol-chloroform.

3. In vitro transcription of T7-sgRNA-miR-382/miR-154

Take 1 μg of each linearized plasmid (T7-sgRNA-miR-382, T7-sgRNA-miR-154), use an in vitro transcription kit (ambion AM1340) for in vitro transcription, and react at 37°C for 3 hours. The specific system is as follows:

4. Use RNA purification kit (ambion AM1908) to purify step 3) in vitro transcription product, the specific system is as follows:

Pass through the adsorption column of the RNA purification kit, wash twice at 12000rpm, 30s, and 70% alcohol, and finally elute with RNase-free water, run gel identification, as shown in Figure 5, the results show that T7-sgRNA-miR-382mRNA and T7-sgRNA were obtained - miR-154 mRNA.

5. Preparation of transgenic mice (time 3 hours)

1) The CRISPR/Cas9 injection system for mice is as follows:

2) injection

Use the Eppendorf 2xTransferMan NK2 microinjector to draw 2 μl of the mixture in step 1) and inject 60 to 80 fertilized eggs.

Five days after the mice were born, the mouse nails were clipped to extract genomic DNA. PCR identification of miR-154, miR-382 genes and sequencing, as shown in Figure 6. The results showed that after miR-382 was knocked out, the mouse heart became smaller, as shown in Figure 7.

Claims (3)

1. A method utilizing CRISPR-Cas9 to specifically knock out the microRNA gene family is characterized in that the method is: (1) taking the px330 plasmid as a template, acting on primer Cas9-R and the primer Cas9-F containing T7 promoter The PCR reaction was carried out under the following conditions, and the PCR reaction product was connected with the pGME-T carrier to obtain the plasmid pGEM-Cas9, and then the plasmid pGEM-Cas9 was linearized, in vitro transcribed and recovered by the RNA purification kit to obtain Cas9 mRNA; (2) px330 plasmid As a template, PCR reaction is carried out under the action of primer sgRNA-R and primer sgRNA-F containing T7 promoter, and then the PCR amplification product is connected to the vector pGEM-T to obtain the vector pGEM-T7-sgRNA; (3) The miR-154sgRNA shown in SEQ ID NO.3 and the miR-382sgRNA shown in SEQ ID NO.4 were respectively connected with the vector pGEM-T7-sgRNA to obtain plasmid T7-sgRNA-miR-382 and plasmid T7-sgRNA-miR- 154, after linearization, in vitro transcription and RNA purification kit recovery, T7-sgRNA-miR-382mRNA and T7-sgRNA-miR-154mRNA were obtained; (4) Cas9mRNA, T7-sgRNA-miR-382mRNA and T7-sgRNA- miR-154mRNA is mixed into a mixture and injected into fertilized mammalian eggs to achieve the purpose of targeted knockout of the microRNA gene; Cas9-F: 5'-TTAATACGACTCACTATAGGATGGACTATAAG GACCACGAC-3'; Cas9-R: 5'-GCGAGCTCTAGGAATTCTTAC-3'; sgRNA-F: 5'-TTAATACGACTCACTATAGGGTGGAAAGGACGAAACA CCGGGTCTTCGAGAAGACCT-3'; sgRNA-R: 5'-AAAAGCACCGACTCGGTGCC-3'. 2. The method according to claim 1, wherein the concentrations of Cas9mRNA, T7-sgRNA-miR-382mRNA and T7-sgRNA-miR-154mRNA are respectively 50ng/μl, 100ng/μl and 10ng/μl. 3. The method according to claim 1, characterized in that the mammalian fertilized eggs are mouse fertilized eggs.