Cell line gene knock-out method for obtaining large fragment deletion through CRISPR/Cas9 system

Abstract

The invention relates to a cell line gene knock-out method for obtaining large fragment deletion through a CRISPR / Cas9 system, and belongs to the field of genetic engineering and genetic modification. A pX458 vector is modified, so that the pX458 vector is provided with DsRed2 and ECFP (Enhanced Cyan Fluorescence Protein); then, a plurality of specific sgRNA sites are designed by aiming at a target gene and are connected into the modified vector; after the cell line transfection, cell groups are sorted by a flow cytometry; single cells subjected to genome editing can be very fast obtained; then, a single-cell DNA (Deoxyribonucleic Acid) sequence is subjected to PCR (Polymerase Chain Reaction) amplification; and single cells with large fragment deletion can be picked out from the single cells through gel electrophoresis. The CRISPR / Cas9 system, the flow cytometry single-cell sorting and fluorescent protein screening on the expression vector are combined, so that the positive monoclonal cells with the large fragment deletion can be obtained in a short time; and the work efficiency of the cell line gene knock-out is greatly improved.

Description

technical field [0001] The invention relates to a cell line gene knockout method for rapidly obtaining large fragment deletion through a CRISPR / Cas9 system, and belongs to the fields of genetic engineering and genetic modification. Background technique [0002] The CRISPR-Cas9 system is the most widely studied and utilized genome editing technology in recent years. Compared with traditional genome editing methods: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), The CRISPR-Cas9 system has the following advantages: simple structure, very easy to transform and operate; high editing efficiency to the genome, easy to obtain gene knockout individuals; no species restrictions in actual use, so it is widely used in animals, plants and It has been widely used in the production of isogene knockout models in cell lines. [0003] Traditional cell line gene knockout requires multiple rounds of resistance screening and dilution after transfectio...

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Problems solved by technology

[0003] Traditional cell line gene knockout requires multiple rounds of resistance screening and dilution after transfection to obtain positive monoclonal cells, which is very time-consuming and laborious, and the false positive rate is high

After the CRISPR / Cas9 system cuts genomic DNA, in most cases, a few bases will be deleted or inserted, and this small change cannot be detected by traditional gel electrophoresis; while other methods, such as: direct Sequencing method, PCR enzyme digestion detection method, T7E1 enzyme digestion detection method, high resolution melting curve detection method can detect whether base changes have occurred at the target site, but cannot obtain the specific number of base deletions or insertions; PAGE Electrophoretic detection method and fluorescent PCR method can obtain detailed information of base changes more accurately, but PAGE electrophoretic detection method is complicated to operate, and fluorescent PCR method requires expensive equipment, which is not suitable for high-throughput screening and large-scale promotion

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