Biomedical Research Support Center (BRSC), Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki, Nagasaki, 852-8523, Japan.
Sci Rep. 2019 Oct 25;9(1):15300. doi: 10.1038/s41598-019-51891-6.
The fine-tuning of gene expression contributes to both basic science and applications. Here, we develop a novel gene expression technology termed CRITGI (CRISPR/Transposon gene integration). CRITGI uses CRISPR/Cas9 to integrate multiple copies of the plasmid pTy1 into Ty1 loci, budding yeast retrotransposons. The pTy1 plasmid harbors a Ty1 consensus sequence for integration, a gene of interest with its own promoter and a selection marker gene. Interestingly, the expression of the pTy1 gene in Ty1 loci could be induced in synthetic complete amino acid depletion medium, which could activate the selection marker gene on pTy1. The induction or repression of the gene on pTy1 depended on Ty1 transcription. Activation of the selection marker gene on pTy1 triggered Ty1 transcription, which led to induction of the gene on pTy1. The gene on pTy1 was not transcribed with Ty1 mRNA; the transcription required its own promoter. Furthermore, the trimethylation of histone H3 on lysine 4, a landmark of transcriptionally active chromatin, accumulated at the 5' end of the gene on pTy1 following selection marker gene activation. Thus, CRITGI is a unique gene regulation system to induce the genes on pTy1 in amino acid depletion medium and utilizes Ty1 transcription to create a chromatin environment favorable for the transcription of the genes on pTy1.
基因表达的微调有助于基础科学和应用。在这里，我们开发了一种新的基因表达技术，称为 CRITGI（CRISPR/转座子基因整合）。CRITGI 使用 CRISPR/Cas9 将多个质粒 pTy1 整合到 Ty1 基因座中，这是一种芽殖酵母逆转录转座子。pTy1 质粒携带用于整合的 Ty1 共有序列、带有自身启动子和选择标记基因的目的基因。有趣的是，在合成完全氨基酸耗尽培养基中可以诱导 Ty1 基因座中 pTy1 基因的表达，这可以激活 pTy1 上的选择标记基因。pTy1 上基因的诱导或抑制取决于 Ty1 转录。pTy1 上选择标记基因的激活触发 Ty1 转录，从而诱导 pTy1 上基因的表达。pTy1 上的基因不以 Ty1 mRNA 转录；转录需要其自身的启动子。此外，组蛋白 H3 在赖氨酸 4 上的三甲基化，这是转录活性染色质的标志，在选择标记基因激活后，在 pTy1 上的基因 5' 端积累。因此，CRITGI 是一种独特的基因调控系统，可以在氨基酸耗尽培养基中诱导 pTy1 上的基因，并利用 Ty1 转录来创建有利于 pTy1 上基因转录的染色质环境。
