Screening method of gene editing site homozygote without transgenosis

Abstract

The invention relates to the technical field of gene editing, and particularly discloses a screening method of a gene editing site homozygote without transgenosis. Different from the existing screening method, the screening method disclosed by the invention has the advantages that high-throughput screening is carried out on T1-generation individuals subjected to gene editing of a target gene by utilizing a KASP marker primer designed for a transgenic vector, individuals containing transgenic components are removed, then amplification sequencing of a gene editing target section is carried out on non-transgenic individuals, and individuals homozygous at a target gene editing site are screened. The method can be applied to high-throughput identification and screening of various animal and plant gene editing offspring populations, and particularly to large-population editing offsprings, for example, tens of thousands to hundreds of thousands of gene editing individuals need to be identified and screened when a gene editing mutant library is constructed, and the screening efficiency can be remarkably improved by applying the technical scheme provided by the invention.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a method for screening homozygous gene editing sites without transgenes. Background technique [0002] Gene editing technologies include ZFN, TANLEN, CRIPSR and other technologies. CRISPR technology has increasingly become a mainstream technology due to its advantages of simple method and low cost, and has been successfully applied to different species of animals, plants and microorganisms. [0003] Gene editing technology can not only break through the genetic barriers that are difficult to solve in traditional breeding, but also achieve precise changes in specific traits, subverting the existing animal genetic improvement technology path and breeding efficiency. With the continuous improvement of gene editing technology and its wide application in animals and plants, researchers around the world have edited a large number of genomes of different species of plants and anim...

AI Technical Summary

Problems solved by technology

The sequencing of PCR amplification products can be completed by first-generation sequencing or next-generation sequencing. In this system, neither PCR amplification nor first-generation sequencing can achieve high-throughput detection. When the sample volume is large, library construction is the bottleneck that limits the throughput of next-generation sequencing. Higher library fees

[0004] Therefore, the screening and detection of offspring individuals of tens of thousands of gene-edited materials is still labor-intensive and time-consuming. It is urgent to develop a low-cost, high-throughput screening method to make up for the shortcomings of existing technologies.

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