AgoFISH: cost-effective in situ labelling of genomic loci based on DNA-guided dTtAgo protein

DNA fluorescence in situ hybridization (FISH) has been widely used for visualizing spatial localization of genomic regions in eukaryotic nuclei. Current probe preparation methods for FISH involve high probe synthesis cost and complex experimental procedures, thus limiting their applicability. Here, we report a new FISH method named AgoFISH based on the DNA-guided DNA binding activity of the nuclease-deficient Argonaute protein in Thermus thermophilus (dTtAgo). Fluorescently labelled dTtAgo combined with 5′-phosphorylated single-stranded guide DNA can bring the fluorescent label to the endogenous DNA sequence that is complementary to the guide DNA. We demonstrated that AgoFISH can successfully label repetitive sequences in human and mouse cells, including the centromere, the pericentromere and single-copy tandem repeats. Assembling dTtAgo with a designed pool of single-stranded guide DNAs is able to visualize nonrepetitive sequences within coding genes. Furthermore, AgoFISH can also be applied to dual-color labelling without crosstalk between channels. This cost-effective and simple method provides a convenient and powerful tool for chromatin higher-order structure studies as well as FISH-based clinical molecular diagnosis.