Fast detection method of cell line CRISPR/Cas9 gene knock-out

A technology for gene knockout and detection method, applied in the field of genetic engineering, can solve the problems of time-consuming and labor-intensive, expensive enzyme, narrow scope of application, etc., and achieve the effect of saving economy and time cost, high sensitivity and precision, and accurate positioning results.

Inactive Publication Date: 2017-12-05
XINXIANG MEDICAL UNIV
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Problems solved by technology

[0003] After the genomic DNA is cut by the CRISPR/Cas9 system, a few bases are often deleted or inserted at the cutting site, and the difference caused by this change cannot be directly detected by ordinary gel electrophoresis
There are five conventional detection methods: direct sequencing, PCR enzyme digestion detection, T7E1 enzyme digestion detection, PAGE electrophoresis detection, high-resolution melting curve detection (high-resolution melting (HRM) analysis), of which direct sequencing The method can obtain very detailed information about mutants, but its cycle is long, the operation is cumbersome, and cannot be applied on a large scale; the PCR enzyme digestion detection method is extremely dependent on whether there is an available enzyme digestion site near the cleavage site, and the scope of application is very narrow; T7E1 Enzyme digestion detection method is not sensitive enough and requires multiple steps to o

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  • Fast detection method of cell line CRISPR/Cas9 gene knock-out
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  • Fast detection method of cell line CRISPR/Cas9 gene knock-out

Examples

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Example Embodiment

[0053] Example 1

[0054] (1) Design a specific targeting site for the mouse Vav2 gene, and construct a vector, transfect the cell line RAW264.7, and obtain GFP-positive single cells by flow cytometry: the mouse Vav2 gene ID is MGI: 102718 (MGI mouse gene database), the target sites are 5'-GGCCAAGTACTACCGCACCCTGG-3' (SEQ ID NO. 1) and 5'-GTTAGAGATTCAGGAGACCGAGG-3' (SEQ ID NO. 2) in the Exon6 (ENSMUSE00001307648) segment, The vector skeleton uses pX458 (purchased from Addgene). After the vector is constructed, the cell line RAW264.7 is transfected with liposomes. After 40-80 hours, GFP-positive single cells are obtained by flow cytometry (BD fusion) sorting To 96-well cell culture plate;

[0055] (2) Expand the culture of GFP-positive single cells, and take some cells directly through lysis to obtain DNA solution for subsequent PCR amplification: take a small amount of cells (do not require quantification, about 100-1000 is enough), centrifuge at 350g, and centrifuge After 5 minut...

Example Embodiment

[0078] Example 2

[0079] (1) Design a specific targeting site for the mouse Vav1 gene, construct a vector, transfect the cell line B16 and sort by flow cytometry, and finally obtain GFP-positive single cells: mouse Vav1 gene ID is MGI: 98923 (MGI mouse gene database, Transcript ID: ENSMUST00000005889), the target site is (SEQ ID NO.20) in the exon5 (exon ID: ENSMUSE00000138941) section: 5'-CTACGAGGACCTAATGCGCT TGG-3' and (SEQ ID NO. 21): 5'-CGAGGACCTTTATGACTGCG TGG-3', the vector skeleton uses pX458 (purchased from Addgene), after the vector is constructed, the cell line B16 is transfected by liposome, 40-72 hours later, it is passed through a flow cytometer (BDfusion ) Sort the GFP-positive single cells into a 96-well cell culture plate;

[0080] (2) Expand the culture of GFP-positive single cells, and take some cells directly through lysis to obtain DNA solution for subsequent PCR amplification: take a small amount of cells (do not require quantification, about 100-1000 is enou...

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Abstract

The invention relates to a fast detection method of cell line CRISPR/Cas9 gene knock-out, and belongs to the technical field of genetic engineering. The cell genomic DNA (Deoxyribonucleic Acid) can be fast obtained by a direct lysis method; a fluorescence PCR (Polymerase Chain Reaction) technology and a capillary electrophoresis method based on a sequencer are combined; and the large-scale genotype identification can be precisely performed in a short time on monoclonal cells subjected to knock-out of a plurality of gene sites. The use of a kit for extracting the genomic DNA is not needed; the cell treatment time only needs 25 min; the economic cost and the time cost are greatly reduced; the sensitivity and the precision are very high; only a small amount of DNA is needed; and the difference of one basic group can be accurately detected. The DNA extraction is not involved, so that the detection and analysis can be performed through simple dilution, denaturation and capillary electrophoresis after the PCR is performed on a sample; and no complicated steps exist, so that the method is suitable for large-scale mass operation.

Description

technical field [0001] The invention relates to a rapid detection method for cell line CRISPR / Cas9 gene knockout, belonging to the technical field of genetic engineering. Background technique [0002] The CRISPR / Cas9 system is the latest gene-specific editing technology. Because the system is easy to operate, low in cost, and highly efficient in editing genome-specific sites, it is widely used in the fields of animals, plants, and microorganisms. more and more widely. [0003] After the CRISPR / Cas9 system cuts the genomic DNA, a few bases are often deleted or inserted at the cutting site, and the difference caused by this change cannot be directly detected by ordinary gel electrophoresis. There are five conventional detection methods: direct sequencing, PCR enzyme digestion detection, T7E1 enzyme digestion detection, PAGE electrophoresis detection, high-resolution melting curve detection (high-resolution melting (HRM) analysis), of which direct sequencing The method can ob...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2563/107C12Q2545/101
Inventor 卢燎勋张黎琛梁银明黄蓉晁天柱郑前前罗静谷妍蓉袁鹏
Owner XINXIANG MEDICAL UNIV
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