CRISPR/Cas-based mRNA imaging has been developed to labeling of high-abundance mRNAs. A lack of non-genetically encoded mRNA-tagged imaging tools has limited our ability to explore the functional distributions of endogenous low-abundance mRNAs in cells. Here, we developed a CRISPR-Sunspot method based on the SunTag signal amplification system that allows efficient imaging of low-abundance mRNAs with CRISPR/Cas9./nMethods: We created a stable TRE3G-dCas9-EGFP cell line and generated an Inducible dCas9-EGFP imaging system for assessment of two factors, sgRNA and dCas9, which influence imaging quality. Based on SunTag system, we established a CRISPR-Sunspot imaging system for amplifying signals from single-molecule mRNA in live cells. CRISPR-Sunspot was used to track co-localization of Camk2a mRNA with regulatory protein Xlr3b in neurons. CRISPR-Sunspot combined with CRISPRa was used to determine elevated mRNA molecules./nResults: Our results showed that manipulating the expression of fluorescent proteins and sgRNA increased the efficiency of RNA imaging in cells. CRISPR-Sunspot could target endogenous mRNAs in the cytoplasm and amplified signals from single-molecule mRNA. Furthermore, CRISPR-Sunspot was also applied to visualize mRNA distributions with its regulating proteins in neurons. CRISPR-Sunspot detected the co-localization of Camk2a mRNA with overexpressed Xlr3b proteins in the neuronal dendrites. Moreover, we also manipulated CRISPR-Sunspot to detect transcriptional activation of target gene such as HBG1 in live cells./nConclusion: Our findings suggest that CRISPR-Sunspot is a novel applicable imaging tool for visualizing the distributions of low-abundance mRNAs in cells. This study provides a novel strategy to unravel the molecular mechanisms of diseases caused by aberrant mRNA molecules.

中文翻译：

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CRISPR-Sunspot：活细胞中单分子水平内源性低丰度RNA的成像

基于CRISPR / Cas的mRNA成像已被开发用于标记高丰度的mRNA。缺乏非基因编码的带有mRNA标签的成像工具的存在限制了我们探索细胞中内源性低丰度mRNA功能分布的能力。在这里，我们基于SunTag信号放大系统开发了一种CRISPR-Sunspot方法，该方法可使用CRISPR / Cas9 / n对低丰度mRNA进行有效成像。方法：我们创建了稳定的TRE3G-dCas9-EGFP细胞系，并产生了可诱导的dCas9 -EGFP成像系统，用于评估影响成像质量的两个因素，sgRNA和dCas9。基于SunTag系统，我们建立了一个CRISPR-Sunspot成像系统，用于扩增活细胞中单分子mRNA的信号。CRISPR-Sunspot用于跟踪Camk2a的共定位神经元中具有调节蛋白Xlr3b的mRNA。CRISPR-Sunspot与CRISPRa组合用于测定升高的mRNA分子。/n结果：我们的结果表明，操纵荧光蛋白和sgRNA的表达可提高细胞中RNA成像的效率。CRISPR-Sunspot可以靶向细胞质中的内源性mRNA，并从单分子mRNA扩增信号。此外，CRISPR-Sunspot还被用于可视化其在神经元中的调节蛋白的mRNA分布。CRISPR-Sunspot检测到Camk2a mRNA与神经元树突中过表达的Xlr3b蛋白共定位。此外，我们还操纵CRISPR-太阳黑子检测靶基因的转录激活，如HBG1活cells./n结论：我们的发现表明，CRISPR-Sunspot是一种新颖的可用于可视化细胞中低丰度mRNA分布的成像工具。这项研究提供了一种新的策略来阐明异常的mRNA分子引起的疾病的分子机制。