Live-cell RNA imaging using the CRISPR-dCas13 system with modified sgRNAs appended with fluorescent RNA aptamers

Tang, Heng Yuan; Peng, Junran; Peng, Shuang; Wang, Qi; Jiang, Xin; Xue, Xiaocheng; Tao, Yanxin; Xiang, Limin; Ji, Quanjiang; Liu, Song-Mei; Weng, Xiaocheng; Zhou, Xiang

doi:10.1039/d2sc04656c

Cited by 11 publications

(8 citation statements)

References 42 publications

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“…Another modification to the sgRNA involves incorporating arrays of fluorescent RNA aptamers to amplify the signal in the dCas13/sgRNA/FP system, enabling simple and sensitive imaging and tracking of endogenous RNA. ( Tang et al, 2022 ).…”

Section: Fluorescent Signal Amplification Techniquesmentioning

confidence: 99%

Live cell imaging of DNA and RNA with fluorescent signal amplification and background reduction techniques

Song

Hou

Zhang

et al. 2023

Front. Cell Dev. Biol.

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Illuminating DNA and RNA dynamics in live cell can elucidate their life cycle and related biochemical activities. Various protocols have been developed for labeling the regions of interest in DNA and RNA molecules with different types of fluorescent probes. For example, CRISPR-based techniques have been extensively used for imaging genomic loci. However, some DNA and RNA molecules can still be difficult to tag and observe dynamically, such as genomic loci in non-repetitive regions. In this review, we will discuss the toolbox of techniques and methodologies that have been developed for imaging DNA and RNA. We will also introduce optimized systems that provide enhanced signal intensity or low background fluorescence for those difficult-to-tag molecules. These strategies can provide new insights for researchers when designing and using techniques to visualize DNA or RNA molecules.

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Section: Fluorescent Signal Amplification Techniquesmentioning

confidence: 99%

Live cell imaging of DNA and RNA with fluorescent signal amplification and background reduction techniques

Song

Hou

Zhang

et al. 2023

Front. Cell Dev. Biol.

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“…Moreover, simultaneous imaging of multiple targets and multicolor imaging represent future directions for aptamer-based fluorescence imaging. In the context of multicolor imaging, fluorophores can consist of diverse orthogonal aptamer–ligand complexes, 46 fluorescent dyes conjugated to aptamers, 163 or nanomaterials serving as carriers. 23 Our research group has developed an intracellular RNA imaging method based on the CRISPR-dPspCas13b system ( Fig.…”

Section: Modalities Of Aptamer-based Molecular Imaging and Applicationsmentioning

confidence: 99%

Engineered aptamers for molecular imaging

Lin,

Xiao,

Jiang

et al. 2023

Chem. Sci.

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“…A notable feature of the Pepper aptamer is that it can bind to eight different HBC dye analogs to give fluorescence emission across the visible spectrum, ranging from cyan to red . Although some studies have demonstrated the robustness of this aptamer in vitro and in eukaryotic cells, − its activity in different bacteria remains largely unexplored. Only recently, a series of Pepper-based biosensors have been tested in E. coli .…”

Section: Introductionmentioning

confidence: 99%

Investigating the Effect of RNA Scaffolds on the Multicolor Fluorogenic Aptamer Pepper in Different Bacterial Species

Mumbleau,

Chevance,

Hughes

et al. 2024

ACS Synth. Biol.

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RNA synthetic biology tools have primarily been applied in E. coli; however, many other bacteria are of industrial and clinical significance. Thus, the multicolor fluorogenic aptamer Pepper was evaluated in both Grampositive and Gram-negative bacteria. Suitable HBC−Pepper dye pairs were identified that give blue, green, or red fluorescence signals in the E. coli, Bacillus subtilis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Furthermore, we found that different RNA scaffolds have a drastic effect on in vivo fluorescence, which did not correlate with the in vitro folding efficiency. One such scaffold termed DF30−tRNA displays 199-fold greater fluorescence than the Pepper aptamer alone and permits simultaneous dual color imaging in live cells.

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Live-cell RNA imaging using the CRISPR-dCas13 system with modified sgRNAs appended with fluorescent RNA aptamers

Abstract: The development of RNA imaging strategies in live cells is essential to improve our understanding of their role in various cellular functions. We report an efficient RNA imaging method based...

Cited by 11 publications

References 42 publications

Live cell imaging of DNA and RNA with fluorescent signal amplification and background reduction techniques

Live cell imaging of DNA and RNA with fluorescent signal amplification and background reduction techniques

Engineered aptamers for molecular imaging

Investigating the Effect of RNA Scaffolds on the Multicolor Fluorogenic Aptamer Pepper in Different Bacterial Species

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