CRISPR-mediated rapid and effective crop site-specific gene fragment or allele replacement method and system

Abstract

The invention discloses a CRISPR-mediated rapid and effective crop site-specific gene fragment or allele replacement method and system. The system provided by the invention comprises a recombinant vector and a free donor fragment; the recombinant vector comprises a coding sequence of sgRNA1, an expression cassette of a Cas9 gene, a donor fragment and a coding sequence of sgRNA2; the donor fragmentcomprises a target of sgRNA1, a mutant segment and a target of sgRNA2; homologous recombination occurs between the mutant segment and a target segment; and the differences between the mutant segmentand the target segment are as follows: (1) a target nucleotide or fragment is replaced with a mutated nucleotide or fragment, (2) NGG in the target of sgRNA1 is mutated into non-NGG, and (3) the NGG in the target of sgRNA2 is mutated into non-NGG. The invention provides a feasible and effective method for replacing and integrating site-specific gene fragments or alleles for crop breeding improvement, and the method has a great application potential and a wide prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CRISPR-mediated fast and effective crop site-specific gene segment or allele replacement method and system. Background technique [0002] CRISPR / Cas9-mediated genome editing technology has become one of the most powerful tools in molecular biology. Found in bacteria for the first time, it consists of two parts, sgRNA and Cas9. CRISPR / Cas9 causes double-strand breaks (double-strand breaks, DSBs) at the target site through its own endonuclease activity, and then through non-homologous end joining (NHEJ) or homologous recombination Homology-directed repair (HDR) introduces mutations in two ways. Most of the mutations induced by the NHEJ pathway are insertions or deletions of nucleotides, resulting in frameshift mutations, while in HDR, fragment insertions or nucleotide corrections are mediated by homologous donor DNA. The recognition of target sites by the CRISPR / Cas9 s...

AI Technical Summary

Problems solved by technology

The introduction of these genes or excellent traits through conventional hybridization and backcrossing transfer methods requires years of multi-generation material selection, which is time-consuming and laborious

Although CRISPR / Cas9, as a new targeted gene modification technology, has shown broad development potential and application prospects, its application in crop improvement still has certain limitations. HDR occurs particularly infrequently compared to the random repair process of end junctions

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