A method of terminating biallelic transcription

Abstract

The invention provides a method for terminating biallelic gene transcription. The method comprises the steps of 1, building a SA-T2A-antibiotic screening gene-Poly DNA fragment respectively containing two different antibiotic screening genes; 2, designing gRNA according to different targeting genes, and building a Cas9 / gRNA expression vector; 3, inserting the SA-T2A-antibiotic screening gene-Poly DNA fragment obtained in step 1 into the targeting genes, and building homologous recombinant vectors containing the targeting genes; 4, transferring into cells: transfecting the cells with both a pair of homologous recombinant vectors containing the targeting genes obtained in step 3 and the Cas9 / gRNA expression vector containing the targeting genes obtained in step 2; 5, performing screening on the relevant antibiotic of the antibiotic genes adopted in step 1 for at least two weeks to obtain biallelic gene knock-out polyclonal cell lines. The method for terminating biallelic gene transcription can achieve the purpose that when performing inserting into an efficient fixed point, biallelic gene simultaneously knock-out cell lines can be quickly obtained.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for terminating biallelic gene transcription. Background technique [0002] Loss of gene expression plays an important role in studying gene function. The conventional strategy to cause loss of gene expression is RNA interference. However, due to the high off-target rate and incomplete knockout of RNA, the wide application of this method is largely limited. Frameshift mutations of coding genes mediated by gene editing tools, or large fragment deletions targeting entire genes or promoters can effectively achieve the purpose of gene knockout or knockdown, but large fragment deletions may lead to other gene-related functional elements loss, resulting in misjudgment of gene function. In addition, in order to obtain biallelic knockout cell lines in gene knockout, a lot of time is required for monoclonal cell line screening work. [0003] The CRISPR / Cas system is an adapt...

AI Technical Summary

Problems solved by technology

Although this technical solution is theoretically feasible, there may be many problems in actual operation, for example: 1) in mammalian embryonic cells, the incidence of homologous recombination is extremely low, therefore, if relying on the homologous recombination of the cell itself, Even if it can be achieved, allelic targeting is very difficult, and it is even more difficult to simultaneously target biallelic genes in the same cell; compared with traditional embryonic cell gene targeting, terminally differentiated cells are naturally The rate is low, which makes it more difficult to knock out genes relying on conventional homologous recombination; this is also the reason why the patent does not give examples of cell targeting, and the solution has no practical application value; 2) the technical solution does not An example of cell targeting is given. Even if the system uses homologous recombination, it can only be carried out according to traditional gene recombination. The homologous recombination arm of thousands of bp brings great difficulty to the construction of homologous recombination vector ; 3) The system only targets coding genes and does not involve the knockout of non-coding genes

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