Endogenous GFP tagging in the diatom Thalassiosira pseudonana
Endogenous GFP tagging in the diatom Thalassiosira pseudonana
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硅藻假微型海链藻中的内源 GFP 标记
DOI：
10.1101/2022.09.30.510313
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Nam O
中科院分区：
文献类型：
--
作者：
Nam O
蛋白质的调节丰度和空间分布决定了细胞的结构和功能。绿色荧光蛋白（GFP）的发现及其与靶蛋白的融合以确定亚细胞定位的方法彻底改变了细胞生物学。大多数定位研究涉及引入与GFP基因融合的靶基因的额外拷贝，并在组成型启动子的控制下，导致GFP融合蛋白在非天然水平上的表达。在这里，我们开发了一种单一载体CRISPR/Cas9引导的GFP敲入策略，用于在海链藻中进行基因敲入。这使得能够在内源基因组位置精确和无瘢痕地敲入GFP，以在其天然的isandtransregulatory元件下产生GFP融合蛋白，敲入效率超过50%。我们发现，以前未表征的雌激素样蛋白定位于CO2固定蛋白核，并表明通过测量GFP荧光，我们可以跟踪相对蛋白质丰度响应于环境变化。为了实现内源性标记，我们开发了一个金门分子克隆系统，用于快速组装附加体，以转化成海链藻细菌接合。此外，这个多功能的工具箱可以进行CRISPR/Cas9基因编辑，提供广泛的验证荧光团，并使未来在硅藻中进行大规模功能研究成为可能。重要性声明荧光蛋白（FP）标记是一种广泛使用的技术，用于了解蛋白质的空间分布。然而，在随机整合到基因组中的组成型启动子下引入额外的基因拷贝可导致非生物学相关的表达水平、不需要的基因组突变和定位假象。为了克服这一点，我们开发了一种新的单载体系统，能够在全球重要的模型硅藻中进行CRISPR/Cas9引导的内源性GFP标记。这允许在精确的基因组位置处无瘢痕GFP敲入，导致由天然启动子/终止子调节的GFP融合，这有助于准确定位和确定相对蛋白质丰度。此外，开发的模块化克隆框架是用户友好的，并为高通量大规模研究打开了大门，包括FP标记，敲除和敲入。
The regulated abundance and spatial distribution of proteins determines cellular structure and function. The discovery of green fluorescent protein (GFP) and fusing it to a target protein to determine subcellular localization revolutionized cell biology. Most localization studies involve introducing additional copies of a target gene genetically fused to GFP and under the control of a constitutive promoter, resulting in the expression of the GFP-fusion protein at non-native levels. Here we have developed a single vector CRISPR/Cas9 guided GFP knock-in strategy in the diatomThalassiosira pseudonana. This enables precise and scarless knock-in of GFP at the endogenous genomic location to create GFP fusion proteins under their nativecisandtransregulatory elements with knock-in efficiencies of over 50%. We show that a previously uncharacterized bestrophin-like protein localizes to the CO2-fixing pyrenoid and demonstrate that by measuring GFP fluorescence we can track relative protein abundance in response to environmental change. To enable endogenous tagging, we developed a Golden Gate Molecular Cloning system for the rapid assembly of episomes for transformation intoThalassiosira pseudonanavia bacterial conjugation. In addition, this versatile toolbox enables CRISPR/Cas9 gene editing, provides a broad range of validated fluorophores and enables future large-scale functional studies in diatoms.Significance statementFluorescent protein (FP) tagging is a widely utilized technique for understanding the spatial distribution of proteins. However, introducing extra gene copies under constitutive promoters that randomly integrate into the genome can result in non-biologically relevant expression levels, unwanted genomic mutations and localization artefacts. To overcome this, we developed a novel single vector system capable of CRISPR/Cas9-guided endogenous GFP tagging in a globally important model diatom. This allows scarless GFP knock-in at precise genomic locations resulting in GFP fusions regulated by native promoters/terminators, which facilitates accurate localization and determination of relative protein abundance. Moreover, the developed modular cloning framework is user-friendly and opens the door for high throughput large-scale studies, including FP tagging, knock-out, and knock-in.
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影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
16.6
作者：
Karas, Bogumil J.;Diner, Rachel E.;Lefebvre, Stephane C.;McQuaid, Jeff;Phillips, Alex P. R.;Noddings, Chari M.;Brunson, John K.;Valas, Ruben E.;Deerinck, Thomas J.;Jablanovic, Jelena;Gillard, Jeroen T. F.;Beeri, Karen;Ellisman, Mark H.;Glass, John I.;Hutchison, Clyde A., III;Smith, Hamilton O.;Venter, J. Craig;Allen, Andrew E.;Dupont, Christopher L.;Weyman, Philip D.
通讯作者：
Weyman, Philip D.
影响因子：
56.9
作者：
Winzeler, EA;Shoemaker, DD;Davis, RW
通讯作者：
Davis, RW
影响因子：
4.7
作者：
Crozet, Pierre;Navarro, Francisco J.;Lemaire, Stephane D.
通讯作者：
Lemaire, Stephane D.
影响因子：
3.8
作者：
Patel, Satyanarayan;Chauhan, Aditya;Vaish, Rahul
通讯作者：
Vaish, Rahul