A method for terminating lncRNA biallelic transcription

Abstract

The invention provides a method for terminating lncRNA diallele transcription. The method comprises the following steps of 1) constructing CMV-antibiotic screening gene-PolyA DNA fragments containing two different antibiotic screening genes; 2) designing gRNA according to different target genes, and constructing Cas9 / gRNA expression vectors; 3) inserting the CMV-antibiotic screening gene-PolyA DNA fragments obtained in the step 1) into the target genes, and constructing homologous recombinant vectors containing the target genes; 4) introducing cells: co-transfecting the pair of homologous recombinant vectors containing the target genes obtained in the step 3) and the Cas9 / gRNA expression vectors containing the target genes obtained in the step 2) to the cells; and 5) performing screening for at least 2 weeks by using related antibiotics used in the step 1), and obtaining polyclonal cell strains of which lncRNA dialleles are knocked out. According to the method, the purpose of quickly obtaining the cell strains of which the lncRNA dialleles are knocked out at the same time while realizing efficient fixed-point insertion can be achieved.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for terminating long-chain non-coding RNA biallelic gene transcription. Background technique [0002] The study of the function of long non-coding RNA (lncRNA) is becoming a hot spot, and the loss of gene expression plays a very important role in the study of gene function. The conventional strategy to cause loss of gene expression is RNA interference. However, due to the high off-target rate and incomplete down-regulation of RNA, the wide application of this method is largely limited. Although frameshift mutations of coding genes mediated by gene editing tools, or large fragment deletions of entire genes or promoters can effectively achieve the purpose of gene knockout or knockdown. However, frameshift mutations cannot effectively knock out lncRNAs. At the same time, in the study of long non-coding RNA genes, the deletion of large fragments may lead to the loss of ot...

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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