Application of aptamers improves CRISPR-based live imaging of plant telomeres

Abstract

Development of live imaging techniques for providing information how chromatin is organized in living cells is pivotal to decipher the regulation of biological processes. Here, we demonstrate the improvement of a live imaging technique based on CRISPR/Cas9. In this approach, the sgRNA scaffold is fused to RNA aptamers including MS2 and PP7. When the dead Cas9 (dCas9) is co-expressed with chimeric sgRNA, the aptamer-binding proteins fused to fluorescent protein (MCP-FP and PCP-FP) are recruited to the targeted sequence. Compared to previous work with dCas9:GFP, we show that the quality of telomere labelling was improved in transiently transformed Nicotiana benthamiana using aptamer-based CRISPR-imaging constructs. Labelling is influenced by the copy number of aptamers and less by the promoter types. The same constructs were not applicable for labelling of repeats in stably transformed plants and roots. The constant interaction of the RNP complex with its target DNA might interfere with cellular processes. HighlightAptamer-based CRISPR imaging: an opportunity for improving live-cell imaging in plants

Plant phenotyping relevance

植物細胞内のテロメアを可視化するCRISPRライブイメージング手法の改良と比較検証が研究の中心であり、植物の細胞状態を画像取得する方法に該当する。

Code and data availability

The paper describes aptamer-based CRISPR/dCas9 live imaging of plant telomeres with microscopy measurements analyzed in ImageJ and IMARIS. No public phenotype dataset, image collection, author analysis code, or trained model is deposited or referenced with an availability statement. The only URL mentioned (DeskGen) is,

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