CN104894071A - Method for carrying out gene edition on GT1-7 cells - Google Patents

Abstract

The invention relates to a method for gene editing of GT1-7 cells, comprising: laying GT1-7 cells in a 24-well plate; waiting for the cells to adhere to the wall, inserting the 42229 plasmid containing the Cas9 gene and the puromycin resistance gene Transfect into the GT1-7 cell line; when the cells express the puromycin gene, use puromycin to select 42229 to transfect the GT1-7 cells; after screening, remove the medium containing puromycin and replace it with normal high-glucose medium, And expand the culture, until the GT1-7 cells in the culture plate are overgrown, digest and transfer to the culture flask to continue the expansion culture, and the Cas9 stably transfected GT1-7 cells can be obtained. In practical application, the present invention only needs to transfect a specific sgRNA plasmid to perform precise gene editing on the target gene of GT1-7 cells, avoiding the influence of large Cas9 plasmid on transfection efficiency, and has a good application prospect.

Description

A method for gene editing GT1-7 cells

technical field

The invention belongs to the field of CRISPR-Cas9, in particular to a method for gene editing GT1-7 cells.

Background technique

The discovery of the CRISPR sequence can be traced back to 1987. When Nakata and his colleagues were studying the E. coli iap gene, they found that there was a series of 29nt repeating sequences downstream of the gene, and these 29nt repeating elements were replaced by 32nt non- separated by repeating sequences. Over the next decade, as a large number of microbial genes were sequenced, Mojica and his colleagues found that such spaced repeats were widespread in bacteria and archaea [Ishino Y, Shinagawa H, Makino K, et al. sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product[J].Journal of bacteriology,1987,169(12):5429-5433], [Mojica F J M, C, Soria E, et al. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria [J]. Molecular microbiology, 2000, 36(1): 244-246].

In 2002, Jansen and Mojica first used the new term CRISPR to name these regularly spaced repeat sequences. At the same time, Jansen et al. discovered a CRISPR-associated conserved gene family upstream of the CRISPR sequence-Cas[Jansen R, Embden J, Gaastra W, et al. Identification of genes that are associated with DNArepeats in prokaryotes[J].Molecular microbiology, 2002, 43(6):1565-1575]. In 2007, Horvath and his colleagues demonstrated that the spacer sequence in the CRIPSR system acts as a targeting sequence to bind the target DNA and the Cas enzyme mediates the shearing of DNA [Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes[J].Science,2007,315(5819):1709-1712].

According to the different members of the Cas gene family upstream of the CRISPR sequence, the CRISPR system can be roughly divided into three types (type I-III), among which the type II CRISPR system has the least members of the Cas gene family and is the easiest to use as a gene editing tool. 2011 to 2013. Sikney and Cong et al. used the typeII CRISPR-Cas9 system to complete multiple gene editing operations in vitro, in bacteria and in mammals [Sapranauskas R, Gasiunas G, Fremaux C, et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli[ J].Nucleic acids research,2011], [Nucleic acids research,2011; Cong L,Ran F A,Cox D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339(6121):819 -823].

The currently widely used Streptococcus pyogenes type II CRISPR-Cas9 system is mainly composed of three elements: CRISPR, trancrRNA and Cas9. CRISPR elements are located in the downstream part of the gene, while Cas and trancrRNA are located in the upstream part. TrancrRNA and CRISPR are transcribed in different directions, and trancrRNA has two transcription start points. First, CRISPR will be transcribed into a pre-crRNA sequence of 511nt length, and trancrRNA will be transcribed into two primary transcripts of 171nt and 89nt length. The two primary transcripts of trancrRNA have a 25nt sequence that is complementary to the repeat sequence (repeats) of CRISPR. The complementarity of the two enables CRISPR to be processed into a mature crRNA of 39-42nt, while the primary transcript of trancrRNA is processed into a 75nt the sequence of. The 39-42nt mature crRNA contains 20nt spacers, which can complement the target sequence. Cas9 is responsible for double-stranded cutting of the target sequence, and the Cas9 cutting site is usually located 3 nucleotides upstream of the PAM sequence. Among them, the PAM (protospacer adjacent motif) sequence is necessary for the CRISPR system to function [Deltcheva E, Chylinski K, Sharma C M, et al. CRISPR RNAmaturation by trans-encoded small RNA and host factor RNase III[J].Nature,2011 , 471(7340):602-607]. In order to further simplify the CRISPR-Cas9 system, Zhang et al. constructed sgRNA elements for the crRNA:trancrRNA complex, so that only two elements, sgRNA and Cas9, can be used in the CRISPR-Cas9 system to complete precise gene editing without affecting the efficiency of gene editing. Currently, plasmids expressing sgRNA, Cas9 gene, and crRNA, trancrRNA, and Cas9 gene simultaneously can be queried and purchased from the addgene (http://www.addgene.org/) website.

Although CRISPR-Cas9 has been widely used in bacterial, plant, and mammalian cells, it is often cell lines and embryonic cells. For neuronal cells with low transfection efficiency, effective gene editing cannot be formed due to the large size of the Cas9 plasmid. GT1-7 cells are a cell line obtained by transforming mouse GnRH neurons. This cell line retains the phenotype of the original highly differentiated GnRH neurons, can produce neurites and retains the markers of the original neurons, and is an important cell model for the study of GnRH secretion and reproductive development in mice [Mellon PL, Windle J J, Goldsmith P C, et al. Immortalization of hypothalamic GnRH by genetically targeted tumorigenesis[J].Neuron,1990,5(1):1-10].

At present, there are few reports on the use of CRISPR-Cas9 system to study neurons, and there is no report on the use of CRISPR-Cas9 system to study GT1-7 cell lines.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for gene editing of GT1-7 cells. This method only needs to transfect a specific sgRNA plasmid to precisely edit the target gene of GT1-7 cells, avoiding Cas9 Effect of larger plasmids on transfection efficiency. In addition, in practical applications, the use of viruses carrying sgRNA to transfect cells can further improve the efficiency of gene editing, which has a good application prospect.

A method of gene editing GT1-7 cells of the present invention, comprising:

Place GT1-7 cells in a 24-well plate; wait for the cells to adhere to the wall, use the Lipo2000 method to transfect the 42229 plasmid containing the Cas9 gene and the puromycin resistance gene into the GT1-7 cell line; wait for the cells to express puromycin Gene, use puromycin at a final concentration of 0.3ug/ml to screen 42229 transfected GT1-7 cells; after screening, remove the medium containing puromycin, replace it with normal high-glucose medium, and carry out expansion culture, and the culture plate The GT1-7 cells in the medium were overgrown, digested and transferred to a culture flask to continue to expand the culture, and the Cas9 stably transfected GT1-7 cells were obtained.

The specific parameters of the GT1-7 cells being plated in a 24-well plate are 150,000 cells per well, and 6 wells are plated.

The concentration ratios of the 42229 plasmid and Lipo2000 were 500ng/1.5ul, 700ng/1.5ul, 500ng/2.0ul, 700ng/2.0ul and two negative controls, respectively.

The puromycin screening time is 9-10 days.

The present invention obtains Cas9 stably transfected GT1-7 cells through plasmid resistance screening, and can be used in GT1-7 cells for molecular level research in combination with specific sgRNA. In addition, the use of plasmid resistance screening in the present invention is more convenient and faster than PB transposon and viral vector construction of Cas9 stably transfected cells.

Beneficial effect

The present invention provides a method for gene editing GT1-7 cell lines using the CRISPR-Cas9 system. Since the present invention has constructed a Cas9 stably transfected GT1-7 cell line, it only needs to be transfected with a specific sgRNA plasmid in practical applications. Precise gene editing of target genes in GT1-7 cells avoids the impact of larger Cas9 plasmids on transfection efficiency. In addition, in practical applications, the use of viruses carrying sgRNA to transfect cells can further improve the efficiency of gene editing, which has a good application prospect.

Description of drawings

Figure 1 is the result of puromycin screening of GT1-7 cells; wherein, A is a positive clone of GT1-7 cells; B is a negative control;

Figure 2 is the PCR verification of the random integration of the 42229 plasmid into GT1-7 cells; among them, lanes 1-3 are primer 42229-Cas9 positive clones, 4-6 is the mouse genome, 7 is primer 42229-Cas9 PCR negative, and 8-10 is primers 42229-Puro positive clones, 11-13 for mouse genome, 14 for primer 42229-Cas9 PCR negative.

Fig. 3 is the variation of Cas9 gene expression level of positive clone (n=4);

Figure 4 is the electrophoresis image of Cas9 and sgRNA double plasmid transfection GT1-7 cells targeting mir-505 gene T7E1; wherein, lane 1 is Cas9 and sgRNA double plasmid transfection, and lane 2 is untransfected plasmid;

Figure 5 is an electrophoresis image of sgRNA transfected Cas9 stably transfected GT1-7 cells targeting mir-505 gene T7E1; wherein, lane 1 is transfected with 505sgRNA plasmid, and lane 2 is untransfected with 505sgRNA plasmid;

Figure 6 is the sequencing map of Cas9 and sgRNA dual plasmid transfection GT1-7 cells targeting mir-505 gene;

Figure 7 is a sequence diagram of sgRNA transfection Cas9 stably transfected GT1-7 cells targeting mir-505 gene.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

(1) Exploration of puromycin screening for the lowest lethal concentration of GT1-7 cells:

Day 1: Plate GT1-7 cells in a 24-well plate, 60,000 cells per well, and plate 5 wells.

The next day: GT1-7 cells adhere to the wall and grow. Different amounts of puromycin were added to the 5 cell culture wells so that the final puromycin concentrations in the 5 wells were 0.3ug/ml, 0.5ug/ml, 0.7ug/ml, 1ug/ml and 0ug/ml. Afterwards, the lethality of puromycin to the GT1-7 cells in each well was observed every day, and the selection cycle was set to 4 days due to the high toxicity of puromycin.

It was found that the puromycin concentration of 0.3ug/ml could completely kill the cells for 4 days.

(2) Use 0.3ug/ml puromycin to select stable Cas9 gene GT1-7 cells:

Day 1: Plate GT1-7 cells in a 24-well plate, 150,000 cells per well, and plate 6 wells.

The next day: After the cells grow on the wall, use the Lipo2000 method to transfect the 42229 plasmid containing the Cas9 gene and the puromycin resistance gene into the GT1-7 cell line. The concentration ratios of the plasmid and Lipo2000 were: 500ng/1.5ul, 700ng/1.5ul, 500ng/2.0ul, 700ng/2.0ul and two negative controls.

Day 4: After the cells express the puromycin gene, select 42229 transfected GT1-7 cells with puromycin at a final concentration of 0.3ug/ml.

Thirteenth day: After 9 days of selection with a final concentration of 0.3ug/ml puromycin, the culture medium containing puromycin was removed, replaced with a normal high-glucose medium, and expanded culture was carried out (Figure 1). After the GT1-7 cells in the culture plate were overgrown, they were digested and transferred to culture flasks to continue to expand the culture to obtain Cas9 stably transfected GT1-7 cells.

(3) Two pairs of primers were designed to detect whether the 42229 plasmid was randomly integrated into GT1-7 cells. In order to ensure the integration of Cas9 into the cells, one pair of primers was designed on the Cas9 gene, while the other pair of primers was designed on the Puro gene.

PCR reaction system: ddH ₂ O 7.8ul, GT Buffer 1.5ul, dNTP 1.5ul, Taq enzyme 1.5ul, BSA 0.2ul, upstream and downstream primers (2P) 1.5ul, template 1.5ul; total 15.5ul; PCR reaction system: 95 ℃ 2min, (94℃ 30sec, 58℃ 30sec, 72℃ 30s) 35 cycles, 72℃ 10min.

PCR results were identified by 1.5% agarose electrophoresis (Figure 2).

(4) Verify whether the expression of Cas9 in GT1-7 cells stably transfected with the 42229 plasmid is increased from the level of gene expression:

Firstly, the total RNA of GT1-7 cells stably transfected with 42229 plasmid was extracted by Trizol method. Total RNA from GT1-7 was then reverse-transcribed using random primers and oligodT. The Real-time PCR primers are the same as the 42229-Cas9 primers in step (3).

DnaseI treatment system: DnaseI 1ul, 10*buffer 1ul, RNA 1ug, DEPC H ₂ O to 10ul; reaction procedure: 15min at room temperature, 10ul system added 1ul EDTA at 65°C for 10min.

Reverse transcription: System RNA 6.25ul, Random Primer 0.25ul, oligodT 0.25ul, 5*Reaction Buffer 1ul, dNTP mix 1ul, Ribolock RNase Inhibitor 0.5ul, RevertAidReverse Transcriptase 0.5ul, a total of 10.75ul; reaction program: 25°C 5min, 42°C 60min , 70°C for 5min.

cDNA diluted 5 times as a template, Real-time PCR: system DEPC-H ₂ O 4.1ul, 2*Premix 10ul, cDNA 2.5ul, Primer (2P) 3ul, ROX 0.4ul total 20ul; reaction program: 95°C 2min, ( 94°C 30sec, 58°C 20sec, 72°C 30s) 40 cycles.

The results of Real-time PCR were analyzed by 7500 software (Figure 3)

(5) The 41824 plasmid expressing only sgRNA was transfected into GT1-7 cells stably transfected with Cas9.

Day 1: Spread Cas9 stably transfected GT1-7 cells in a 24-well plate, with 200,000 cells per well, and spread 2 wells;

The next day: when the confluence of the cells attached to the wall reached 70-90%, the cells were first starved without serum, and then the 41824 plasmid expressing only sgRNA was transfected into the Cas9 stably transfected GT1-7 cells using the Lipo2000 method. Wherein Lipo2000:41824 plasmid=2.0ul:lug.

When the cells are full, the cells are digested to extract the genomic DNA.

(6) Gene mutation detection: the target gene is amplified by PCR on the cellular genomic DNA obtained in step (5), and then the PCR product is purified. The purified product was detected by T7E1 assay for the mutation of the target gene.

T7E1assay system: PCR product 600ng, buffer 1.1ul, H ₂ O supplemented to 10.5ul; reaction program: 95°C for 10min (95°C cooling by 1°C every 30s) for a total of 70 cycles; 25°C for 1min. Then add 0.45ul T7E1 enzyme and 37°C for 30min.

The result of digestion was detected by 2% agarose gel electrophoresis.

(7) The 41824 plasmid containing only sgRNA targeting the mir-505 gene was transfected into Cas9 stably transfected GT1-7 cells:

The sgRNA target sequence (20nt) is: GTAAATTGATGCACCCAGTG.

Day 1: Spread Cas9 stably transfected GT1-7 cells in a 24-well plate, with 200,000 cells per well, and spread 2 wells;

Day 2: Cells reach 70-90% confluence. Before transfection, the cells were starved for 2 hours with serum-free medium, and then the lipo2000 method was used to transfect the 41824 plasmid into the cells, Lipo2000:41824 plasmid=2.0ul:lug. Six hours after transfection, cells were cultured with normal medium instead of serum-free medium.

Day 5: 3 days after transfection, the cells filled the wells, and the genome of the cells was extracted using the Biotech Animal Genome Extraction Kit.

(8) Carry out PCR amplification for mouse mir-505 gene:

Mir-505-F:catgccaacaaacccagtga 20bp

Mir-505-L: ctggcttctactccctggtg 20bp product size 942bp

PCR reaction system: H ₂ O 3.725ul, buffer 3ul, upstream and downstream primers (2P) 3ul, dNTP 1.5ul, Mg ²⁺ 1.2ul, Taq enzyme 0.075ul, template 2.5ul; reaction program: 95°C 15min, (94°C 30sec, 62°C 30sec, 72°C 1min) 40 cycles, 72°C 5min.

PCR results were detected by 1.5% agarose gel electrophoresis.

(9) Use the Sangon PCR Product Purification Kit to purify and recover the PCR product in step (2), and then perform T7E1 assay to detect whether there is a mutation in the mir-505 gene:

T7E1assay system: PCR product 600ng, buffer 1.1ul, H ₂ O supplemented to 10.5ul; reaction program: 95°C for 10min (95°C cooling by 1°C every 30s) for a total of 70 cycles; 25°C for 1min. Then add 0.45ul T7E1 enzyme, 37°C for 30min.

The results of enzyme digestion were detected by 2.0% agarose gel electrophoresis. Compared with the electrophoresis results of transfection of GT1-7 cells using Cas9 and sgRNA dual plasmid system before, there was no enzyme digestion band (Figure 4), the gene editing method used in the present invention A clear enzyme-cut band can be seen, and the enzyme-cut band is about two bands of 400bp and 500bp (Figure 5).

(10) The PCR purified product of the mir-505 gene was sent to Sangon for sequencing, compared with the previous results of transfection of GT1-7 cells using the dual plasmid system of Cas9 and sgRNA (Figure 6). The gene editing method adopted in the present invention can be seen in the sequencing map where disordered peaks appear, and the position of the disordered peaks starts near 3 bp upstream of the PAM sequence ( FIG. 7 ). This result demonstrates the effectiveness of this gene editing method for GT1-7 cells.

Claims (4)

1. A method for gene editing GT1-7 cells, comprising: Place GT1-7 cells in a 24-well plate; wait for the cells to adhere to the wall, use the Lipo2000 method to transfect the 42229 plasmid containing the Cas9 gene and the puromycin resistance gene into the GT1-7 cell line; wait for the cells to express puromycin Gene, use puromycin at a final concentration of 0.3ug/ml to screen 42229 transfected GT1-7 cells; after screening, remove the medium containing puromycin, replace it with normal high-glucose medium, and carry out expansion culture, and the culture plate The GT1-7 cells in the medium were overgrown, digested and transferred to a culture flask to continue to expand the culture, and the Cas9 stably transfected GT1-7 cells were obtained. 2. A method for gene editing GT1-7 cells according to claim 1, characterized in that: said GT1-7 cells are plated on a 24-well plate, and the specific parameters are 150,000 cells per well, and 6 holes. 3. A method for gene editing GT1-7 cells according to claim 1, characterized in that: the concentration ratio of the 42229 plasmid and Lipo2000 is 500ng/1.5ul, 700ng/1.5ul, 500ng/ 2.0ul, 700ng/2.0ul and two negative controls. 4. A method for gene editing GT1-7 cells according to claim 1, characterized in that: the puromycin selection time is 9-10 days.