We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe 'glonads', a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663 .

中文翻译：

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Split-wrmScarlet和split-sfGFP：秀丽隐杆线虫内源性蛋白质的更快，更轻松的荧光标记工具

我们创建并共享一个新的红色荧光团，以及一系列菌株，试剂和规程，以使其更快，更轻松地用荧光标签标记内源秀丽隐杆线虫蛋白。CRISPR介导的秀丽隐杆线虫蛋白质荧光标记是一种宝贵的工具，但是插入荧光团大小的DNA片段比进行小的基因编辑要困难得多。原则上，高亲和力不对称分裂的荧光蛋白可以解决秀丽隐杆线虫的这一问题：小片段可以快速，轻松地融合到几乎任何目标蛋白质上，并且可以在大片段表达和互补的任何位置进行检测。但是，目前只有一种可用的菌株稳定表达分裂的荧光蛋白的大片段，从而将该溶液限制在高度自发荧光的绿色通道中的单个组织（种系）中。没有可用的秀丽隐杆线虫系表达分裂的红色荧光蛋白的未结合的大片段，与标准的分裂的sfGFP蛋白相比，即使是最先进的分裂的红色荧光蛋白也很暗。在这项研究中，我们设计了一种明亮的，高亲和力的新拆分红色荧光团split-wrmScarlet。我们产生转基因秀丽隐杆线虫可以方便地在肌肉或生殖细胞中进行单色标记，并在体细胞中进行双色标记。我们还描述了“ glonads”，这是种系的一种新型表达策略，而传统的表达策略却在其中挣扎。我们通过将split-wrmScarlet靶向其产物标记不同细胞器的几个基因来验证这些菌株，并提供了易于，无克隆的CRISPR / Cas9编辑的方案。随着在不同组织或细胞器中标记的FP分离菌株的集合的扩大，我们将在doi.org/10.5281/zenodo.3993663上发布更新。