Double-donor mediated drug/fluorescence collaborative screening-based diallelic editing system

Abstract

The invention discloses a double-donor mediated drug / fluorescence collaborative screening-based diallelic editing system. The system comprises two donor carriers, the donor vector sequence comprises aleft target gene homologous sequence, a right target gene homologous sequence, a point mutation site and an exogenous sequence located between the left target gene homologous sequence and the right target gene homologous sequence. The exogenous sequences are divided into a plurality of expression cassettes with thymokinase screening genes, green fluorescent proteins and puromycin resistance genesand a plurality of expression cassettes with thymokinase screening genes, red fluorescent proteins and bleomycin resistance genes. The gene editing system constructed by the invention can be used forcarrying out accurate and efficient diallelic editing on the genome.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a gene editing system based on a double-strand break repair mechanism, in particular to introducing mutations and exogenous screening genes into two biallelic loci when the genome is repaired by homologous recombination to obtain stable Inherited transgenic cell lines. Background technique [0002] Genome editing technology relies on artificial specific nucleases to create double-strand breaks on the genome and the resulting DNA repair pathways to achieve gene editing. Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated genes (CRISPR / Cas) system and the previous two generations rely on the artificial sequence specificity of protein module and nucleotide pairing recognition Compared with nuclease (ZFN and TALEN) systems, the CRISPR / Cas system relies on pair recognition between nucleotides and has obvious advantages in gene editing. Among them, t...

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Problems solved by technology

However, at present, one kind of donor is mainly used to edit the target genes of the two sister chromosomes, resulting in three kinds of gene-edited cells with different repair results after repairing by the cell's own double-strand break repair mechanism: 1) Two sister chromosomes Cells that have been repaired by the NHEJ pathway; 2) monoallelic edited cells with one sister chromosome repaired by NHEJ and the other sister chromosome with HDR repair; 3) biallelic edited cells with both sister chromosomes repaired by the HDR pathway

[0004] Existing donor strategies are less efficient in obtaining biallelic homozygotes (for example, Chinese patent CN 106191116 A, CRISPR / Cas9-based exogenous gene knock-in integration system and its establishment method and application, Zhang Zhiying, etc.), urgently needed Development of a more efficient biallelic gene editing system

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