mScarlet and split fluorophore mScarlet resources for plasmid-based CRISPR/Cas9 knock-in in C. elegans.
mScarlet and split fluorophore mScarlet resources for plasmid-based CRISPR/Cas9 knock-in in C. elegans.
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DOI：
10.17912/micropub.biology.000871
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发表时间：
2023
影响因子：
--
通讯作者：
Doonan, Ryan
中科院分区：
文献类型：
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作者：
Witten, Gillian;DeMott, Ella;Huang, George;Zelasko, Francis;de Jesus, Bailey;Mulchand, Chandi;Schuck, Liam;Pullman, Stephen;Perez, Amelie;Mahableshwarkar, Priya;Wu, Zheng;Cardona, Eric Andrew;Pierce, Jonathan T;Dickinson, Daniel J;Doonan, Ryan
荧光蛋白允许在活体动物中观察基因的表达及其蛋白质产物的行为。通过 CRISPR 基因组工程创建内源荧光蛋白标签的能力彻底改变了这种表达的真实性，mScarlet 目前是我们用于可视化体内基因表达的首选红色荧光蛋白 (RFP)。在这里，我们将先前针对线虫优化的 mScarlet 版本和分裂荧光团 mScarlet 克隆到基于 SEC 的质粒系统中，以进行 CRISPR/Cas9 敲入。理想情况下，内源标签易于可见，同时不会干扰目标蛋白的正常表达和功能。对于荧光蛋白标签（例如 GFP 或 mCherry）大小的一小部分的低分子量蛋白质和/或已知以这种方式标记时无功能的蛋白质，分裂荧光团标记可能是一种替代方法。在这里，我们使用 CRISPR/Cas9 敲入，用分裂荧光团 wrmScarlet 标记三种此类蛋白质：HIS-72、EGL-1 和 PTL-1。尽管我们发现分裂荧光团标签不会破坏任何这些蛋白质的功能，但不幸的是我们无法用落射荧光观察大多数这些标签的表达，这表明分裂荧光团标签作为内源报告基因通常非常有限。尽管如此，我们的质粒工具包提供了一种新资源，可以在秀丽隐杆线虫中直接敲入 mScarlet 或分裂的 mScarlet。
Fluorescent proteins allow the expression of a gene and the behavior of its protein product to be observed in living animals. The ability to create endogenous fluorescent protein tags via CRISPR genome engineering has revolutionized the authenticity of this expression, and mScarlet is currently our first-choice red fluorescent protein (RFP) for visualizing gene expression in vivo . Here, we have cloned versions of mScarlet and split fluorophore mScarlet previously optimized for C. elegans into the SEC-based system of plasmids for CRISPR/Cas9 knock-in. Ideally, an endogenous tag will be easily visible while not interfering with the normal expression and function of the targeted protein. For low molecular weight proteins that are a fraction of the size of a fluorescent protein tag (e.g. GFP or mCherry) and/or proteins known to be non-functional when tagged in this way, split fluorophore tagging could be an alternative. Here, we used CRISPR/Cas9 knock-in to tag three such proteins with split-fluorophore wrmScarlet: HIS-72, EGL-1, and PTL-1. Although we find that split fluorophore tagging does not disrupt the function of any of these proteins, we were unfortunately unable to observe the expression of most of these tags with epifluorescence, suggesting that split fluorophore tags are often very limited as endogenous reporters. Nevertheless, our plasmid toolkit provides a new resource that enables straightforward knock-in of either mScarlet or split mScarlet in C. elegans.