Efficient targeted mutation of genomic essential genes in yeast Saccharomyces cerevisiae.

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ID: 94783
2020
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Abstract
Targeted gene mutation by allelic replacement is important for functional genomic analysis and metabolic engineering. However, it is challenging in mutating the essential genes with the traditional method by using a selection marker, since the first step of essential gene knockout will result in a lethal phenotype. Here, we developed a two-end selection marker (Two-ESM) method for site-directed mutation of essential genes in Saccharomyces cerevisiae with the aid of the CRISPR/Cas9 system. With this method, single and double mutations of the essential gene ERG20 (encoding farnesyl diphosphate synthase) in S. cerevisiae were successfully constructed with high efficiencies of 100%. In addition, the Two-ESM method significantly improved the mutation efficiency and simplified the genetic manipulation procedure compared with traditional methods. The genome integration and mutation efficiencies were further improved by dynamic regulation of mutant gene expression and optimization of the integration modules. This Two-ESM method will facilitate the construction of genomic mutations of essential genes for functional genomic analysis and metabolic flux regulation in yeasts. KEY POINTS: • A Two-ESM strategy achieves mutations of essential genes with high efficiency of 100%. • The optimized three-module method improves the integration efficiency by more than three times. • This method will facilitate the functional genomic analysis and metabolic flux regulation.
Reference Key
yang2020efficientapplied Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Yang, Shan;Cao, Xuan;Yu, Wei;Li, Shengying;Zhou, Yongjin J;
Journal Applied microbiology and biotechnology
Year 2020
DOI
10.1007/s00253-020-10405-5
URL
Keywords
genome editing yeast crispr/cas9 essential gene site-directed mutation

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