Saccharomyces cerevisiae large-fragment gene editing method

Abstract

The invention discloses a saccharomyces cerevisiae large-fragment gene editing method. The editing method comprises the following steps: (1) designing a GIM gene expression cassette, specifically, amplifying an I-SceI gene expression cassette controlled by a galactose promoter from a plasmid pGSHU, inserting an I-SceI recognition sequence into each of an upstream primer and a downstream primer, and enabling a homologous arm to be 52bp; (2) constructing a double-DSBs mutant strain, specifically, transforming into yeast by utilizing a PEG / LiAC method, coating on a Hyg screening plate to grow, selecting a grown monoclonal strain, extracting a genome, and verifying; and (3) designing a target gene expression cassette, specifically, connecting a target gene with a KanMX6 selection marker through overlap extension PCR, and obtaining an upstream and downstream homologous arm 68bp which is a base sequence at two ends of upstream DSB in the double DSBs. According to the saccharomyces cerevisiae large-fragment gene editing method, the knock-in of a large-fragment gene becomes possible, the knock-in site is controllable, meanwhile the probability of an off-target phenomenon is reduced, and the accuracy is improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a method for deleting a large segment of DNA sequence or integrating a large segment of exogenous gene in Saccharomyces cerevisiae. Background technique [0002] Gene editing is a biotechnology that deletes, knocks in or replaces target genes in specific microorganisms / cells to obtain new functions or phenotypes. It is the foundation of basic research and modern food, drug, biochemical and other industries. It has a wide range of applications in the fields of industrial microbial strain transformation and disease treatment. Because of its specificity and high efficiency, it has gradually become the focus of scientific research in recent years. [0003] At a specific part of DNA, when a DNA double strand break (Double Strand Breaks, DSB) occurs, it can recombine with a DNA fragment containing a homologous sequence, resulting in a change in the gene sequence or the integr...

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Problems solved by technology

[0008] The CRISPR / Cas system has a short cycle and low cost, but there are still some disadvantages: the CRISPR / Cas system may perform nucleic acid cleavage at unintended sites, resulting in off-target effects, thereby increasing the risk of unknown recombination

[0010] Through the search, no patent publications related to the patent application of the present invention have been found

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