CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations in place, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold for the endogenous N. benthamiana DFR gene, with negligible expression in the absence of the trigger. The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.

中文翻译：

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用于诱导基于 CRISPR/Cas9 的转录激活的铜开关严密调节本氏烟草中的基因表达

基于 CRISPR 的可编程转录激活剂 (PTA) 用于植物重新连接基因网络。以时间和剂量依赖性方式更好地调整它们的活性应该可以精确控制基因表达。在这里，我们报告了一种名为 CI-switch 的铜诱导系统的优化，用于本氏烟草中的条件基因激活. 在铜存在的情况下，铜反应因子 CUP2 发生构象变化并结合名为铜结合位点 (CBS) 的 DNA 基序。在这项研究中，我们测试了几个与 CUP2 融合的激活域，发现非病毒 Gal4 域导致配备最小启动子的报告基因的强烈激活，提供优于以前设计的优势。为了将铜调节与下游可编程元件连接起来，我们分析了强 dCasEV2.1 PTA 的几种铜依赖性配置，旨在最大化激活范围，同时最小化不需要的背景表达。最佳配置包括对 PTA 的两种蛋白质成分（即 dCas9:EDLL 和 MS2:VPR）的双铜调节，以及第三种成分的组成型 RNA pol III 驱动表达，带有 MS2 RNA 结合域锚定位点的引导 RNA。有了这些优化，CI/dCasEV2.1 系统使内源性的铜依赖性激活率提高了 2,600 倍。本氏烟草DFR 基因，在没有触发器的情况下表达可忽略不计。铜对 CI/dCasEV2.1 的严格调控使该系统成为有条件生产植物源性代谢物和现场重组蛋白的理想选择。