On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members.
On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members.
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用DEXCON，DEXOGRON和LUXON对内源基因的按需表达控制揭示了Rab11家族成员的差异动力学。
DOI：
10.7554/elife.76651
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发表时间：
2022-06-16
期刊：
影响因子：
7.7
通讯作者：
Caswell, Patrick T.
中科院分区：
文献类型：
--
作者：
Gemperle, Jakub;Harrison, Thomas S.;Flett, Chloe;Adamson, Antony D.;Caswell, Patrick T.
CRISPR技术使基因敲除的产生在细胞中广泛实现。然而，一旦失活，它们的重新激活仍然很困难，特别是在二倍体细胞中。在这里，我们提出了DexCon(多西环素介导的内源基因表达控制)、DExogron(DexCon结合生长素介导的靶向蛋白降解)和LUXON(光响应DExCon)方法，它们结合了CRISPR-Cas9介导的一步靶向敲打荧光蛋白和先进的Tet诱导的TRE3GS启动子。这些方法将阻断活跃的基因表达与按需重新激活表达的能力相结合，包括激活沉默的基因。系统的控制可以使用多西环素或时空光，我们展示了在密切相关的囊泡运输调节因子Rab11家族中的功能敲除/拯救。荧光蛋白敲入通过单等位基因修饰产生与微光活显微镜兼容的明亮信号，有可能同时对同一基因的不同等位基因进行成像，并绕过了使用克隆的需要。蛋白质水平很容易通过细胞分选(DExCon)、光照时间(Luxon)或将细胞暴露于不同水平的生长素(DExogron)来调节以与内源表达相对应。此外，我们的方法使我们能够量化先前未预见到的差异，包括高度相似的内源性Rab11家族成员在囊泡动力学、转铁蛋白受体循环、表达动力学和蛋白质稳定性方面的差异，以及它们在三重敲入卵巢癌细胞系中的共存。
CRISPR technology has made generation of gene knock-outs widely achievable in cells. However, once inactivated, their re-activation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation), and LUXon (light responsive DExCon) approaches which combine one-step CRISPR-Cas9-mediated targeted knockin of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene expression with the ability to re-activate expression on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knock-out/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene, and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon), or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, transferrin receptor recycling, expression kinetics, and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in ovarian cancer cell lines.