基于 CRISPR-Cas9 的内源性组蛋白 H3 和 H4 组合突变 shuffling 系统。
A CRISPR-Cas9 based shuffle system for endogenous histone H3 and H4 combinatorial mutagenesis.
机构信息
Laboratory of Feed Biotechnology, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, College of Veterinary Medicine, China Agricultural University, Haidian District, Beijing, 100193, China.
School of Life Sciences, Chongqing University, No. 55 Daxuecheng South Rd., Shapingba, Chongqing, 401331, China.
出版信息
Sci Rep. 2021 Feb 8;11(1):3298. doi: 10.1038/s41598-021-82774-4.
Post-translational modifications of histone proteins greatly impact gene expression and cell fate decisions in eukaryotes. To study these, it is important to develop a convenient, multiplex, and efficient method to precisely introduce mutations to histones. Because eukaryotic cells usually contain multiple copies of histone genes, it is a challenge to mutate all histones at the same time by the traditional homologous recombination method. Here, we developed a CRISPR-Cas9 based shuffle system in Saccharomyces cerevisiae, to generate point mutations on both endogenous histone H3 and H4 genes in a rapid, seamless and multiplex fashion. Using this method, we generated yeast strains containing histone triple H3-K4R-K36R-K79R mutants and histone combinatorial H3-K56Q-H4-K59A double mutants with high efficiencies (70-80%). This CRISPR-Cas9 based mutagenesis system could be an invaluable tool to the epigenetics field.
组蛋白蛋白的翻译后修饰极大地影响真核生物中的基因表达和细胞命运决定。为了研究这些，开发一种方便、多重和高效的方法来精确地引入组蛋白突变是很重要的。由于真核细胞通常含有多个组蛋白基因的副本，因此通过传统的同源重组方法同时突变所有组蛋白是一项挑战。在这里，我们在酿酒酵母中开发了一种基于 CRISPR-Cas9 的改组系统，以快速、无缝和多重的方式在内源性组蛋白 H3 和 H4 基因上产生点突变。使用这种方法，我们以 70-80%的高效率生成了含有组蛋白三突变 H3-K4R-K36R-K79R 突变体和组蛋白组合 H3-K56Q-H4-K59A 双突变体的酵母菌株。这种基于 CRISPR-Cas9 的诱变系统可能是表观遗传学领域的一个非常有价值的工具。
Zotero 插件