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的内源性低丰度 RNA 成像


基于 CRISPR/Cas 的 mRNA 成像已被开发用于标记高丰度 mRNA。缺乏非基因编码的 mRNA 标记成像工具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，我们开发了一种基于 SunTag 信号放大系统的 CRISPR-Sunspot 方法，该方法允许使用 CRISPR/Cas9 对低丰度 mRNA 进行有效成像。/n方法：我们创建了稳定的 TRE3G-dCas9-EGFP 细胞系并生成了诱导型 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CRISPR-Sunspot成像系统，用于放大活细胞中单分子mRNA的信号。 CRISPR-Sunspot 用于追踪神经元中Camk2a mRNA 与调节蛋白 Xlr3b 的共定位。 CRISPR-Sunspot 与 CRISPRa 结合用于确定升高的 mRNA 分子。/n结果：我们的结果表明，操纵荧光蛋白和 sgRNA 的表达提高了细胞中 RNA 成像的效率。 CRISPR-Sunspot 可以靶向细胞质中的内源 mRNA，并放大单分子 mRNA 的信号。此外，CRISPR-Sunspot 还被应用于神经元中 mRNA 分布及其调节蛋白的可视化。 CRISPR-Sunspot 检测到Camk2a mRNA 与神经元树突中过度表达的 Xlr3b 蛋白的共定位。此外，我们还操纵 CRISPR-Sunspot 来检测活细胞中HBG1等靶基因的转录激活。/n结论：我们的研究结果表明 CRISPR-Sunspot 是一种新型适用的成像工具，用于可视化细胞中低丰度 mRNA 的分布。 这项研究提供了一种新的策略来揭示异常 mRNA 分子引起的疾病的分子机制。