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bioRxiv · 10.1101/2020.08.25.265306

CAS-LiveFISH: Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos

Pertsinidis, A.·Geng, Y.
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Abstract

Visualizing the 4D genome in live cells is essential for understanding its regulation. Programmable DNA-binding probes, such as fluorescent clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector (TALE) proteins have recently emerged as powerful tools for imaging specific genomic loci in live cells. However, many such systems rely on genetically-encoded components, often requiring multiple constructs that each must be separately optimized, thus limiting their use. Here we develop efficient and versatile systems, based on in vitro transcribed single-guide-RNAs (sgRNAs) and fluorescently-tagged recombinant, catalytically-inactivated Cas9 (dCas9) proteins. Controlled cell delivery of pre-assembled dCas9-sgRNA ribonucleoprotein (RNP) complexes enables robust genomic imaging in live cells and in early mouse embryos. We further demonstrate multiplex tagging of up to 3 genes, tracking detailed movements of chromatin segments and imaging spatial relationships between a distal enhancer and a target gene, with nanometer resolution in live cells. This simple and effective approach should facilitate visualizing chromatin dynamics and nuclear architecture in various living systems.

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Pertsinidis, A., Geng, Y.. 2020-08-25. CAS-LiveFISH: Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos. https://doi.org/10.1101/2020.08.25.265306

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