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

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...

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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 operate, and T7E1 enzyme is expensive, which is not suitable for large-scale screening; PAGE electrophoresis detection method is cumbersome, expensive reagents, time-consuming and laborious; high-resolution melting curve detection method only It can detect whether there are mutations, but cannot get the specific number of base insertions or deletions; therefore, none of the above methods can quickly and accurately detect mutants at the same time

[0004] The Chinese invention patent with the publication number CN105177126A discloses a method for rapidly detecting gene knockout mice, which uses fluorescent PCR technology combined with standard molecular weight internal references to quickly detect mouse knockout genes, but only uses less dense internal reference molecular weights to detect The results were imprecise, the assay was limited to mice, and the technique was not applied to the detection of gene knockouts in cell lines

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