A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana.
A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana.
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DOI：
10.1186/s12896-022-00741-x
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发表时间：
2022-03-24
影响因子：
3.5
通讯作者：
Vazquez-Vilar M
中科院分区：
文献类型：
--
作者：
Garcia-Perez E;Diego-Martin B;Quijano-Rubio A;Moreno-Giménez E;Selma S;Orzaez D;Vazquez-Vilar M
基于crispr的可编程转录激活子（PTAs）用于植物基因网络的重新布线。以时间和剂量依赖的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，我们报道了一种铜诱导系统的优化，称为ci开关，用于烟叶的条件基因激活。在铜存在的情况下，铜响应因子CUP2发生构象变化并结合一个名为铜结合位点（copper-binding site， CBS）的DNA基序。在这项研究中，我们测试了几种与CUP2融合的激活结构域，发现非病毒Gal4结构域导致配备最小启动子的报告基因的强烈激活，比以前的设计提供了优势。为了将铜调控与下游可编程元件连接起来，我们分析了强dCasEV2.1 PTA的几种铜依赖配置，旨在最大化激活范围，同时最小化不希望的背景表达。最佳配置包括双铜调控PTA的两种蛋白质成分，即dCas9:EDLL和MS2:VPR，以及组成性RNA pol iii驱动的第三种成分的表达，这是一种具有MS2 RNA结合域锚定位点的引导RNA。通过这些优化，CI/dCasEV2.1系统导致内源性benthamiana DFR和PAL2基因的铜依赖激活率分别为2600倍和245倍，在没有触发器的情况下可以忽略表达。铜在CI/dCasEV2.1上的严格调控使该系统非常适合有条件地在田间生产植物衍生代谢物和重组蛋白。在线版本包含补充材料，可在10.1186/s12896-022-00741-x获得。
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 programmable 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, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, 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. The online version contains supplementary material available at 10.1186/s12896-022-00741-x.
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影响因子：
48
作者：
Mali, Prashant;Esvelt, Kevin M.;Church, George M.
通讯作者：
Church, George M.
影响因子：
64.8
作者：
Konermann S;Brigham MD;Trevino AE;Joung J;Abudayyeh OO;Barcena C;Hsu PD;Habib N;Gootenberg JS;Nishimasu H;Nureki O;Zhang F
通讯作者：
Zhang F
影响因子：
4.3
作者：
Olsen, Kristine M.;Lea, Unni S.;Lillo, Cathrine
通讯作者：
Lillo, Cathrine
影响因子：
7.4
作者：
Andres, Jennifer;Blomeier, Tim;Zurbriggen, Matias D.
通讯作者：
Zurbriggen, Matias D.
影响因子：
4.3
作者：
Granger, CL;Cyr, RJ
通讯作者：
Cyr, RJ