Suicide-homicide plasmid, saccharomyces cerevisiae traceless gene editing method using suicide-homicide plasmid and application

Abstract

The invention provides a suicide-homicide plasmid, a saccharomyces cerevisiae traceless gene editing method using the suicide-homicide plasmid and application. The suicide-homicide plasmid comprises areplication origin, a selection marker, a Cas9 protein expression cassette and a sgRNA1 expression cassette, wherein the sgRNA1 expression cassette comprises a promoter, sgRNA1, scaffold and a termination sequence; and the sgRNA1, the scaffold and the termination sequence are designed for a common sequence of a gene editing vector and the suicide-homicide plasmid. The gene editing vector is transferred into saccharomyces cerevisiae, and then the suicide-homicide plasmid is transferred into the saccharomyces cerevisiae, so that a traceless edited saccharomyces cerevisiae strain can be obtained. The saccharomyces cerevisiae is edited by using the suicide-homicide plasmid, so that selection of the strain which successfully transforms the gene editing plasmid after gene editing is not affected, and the plasmid eliminating success rate is remarkably increased; and meanwhile, the traceless editing effect can be achieved through two rounds of plasmid transformation, and the traceless editingrate of the saccharomyces cerevisiae is further increased.

Description

technical field [0001] The present invention relates to the CRISPR / Cas9 technology in the field of gene editing technology, in particular to a method and application of Saccharomyces cerevisiae traceless gene editing, and in particular to a suicide-homicidal plasmid, and the method and application of Saccharomyces cerevisiae traceless gene editing. Background technique [0002] Once CRISPR / Cas9 technology came out, it became one of the focuses of the world. CRISPR / Cas9 technology mainly consists of two elements: the Cas9 protein that cuts the DNA strand and the gRNA that recognizes the target site. The specific gRNA guides the Cas9 protein to cut the DNA double strand at the target site, resulting in a DNA double strand break. Then the cell repairs the broken DNA through its own repair mechanism: homologous recombination system (HR) and non-homologous end-joining pathway (NHEJ), realizing gene knockout and insertion, and achieving the purpose of gene editing. CRISPR / Cas9 g...

AI Technical Summary

Problems solved by technology

However, when using this method, the plasmid may be excised before successful gene editing, so that Cas9 protein and gRNA cannot continue to be expressed, and the effect of gene editing cannot be achieved

Moreover, even if this method has successfully carried out traceless gene editing, because the plasmid has been eliminated and the selection marker has been lost, it is difficult to screen out the S.

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