CRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci with one guide RNA per locus.
Patricia A ClowMenghan DuNathaniel JilletteAziz TaghbaloutJacqueline Jufen ZhuAlbert Wu ChengPublished in: Nature communications (2022)
Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and are ineffective in labeling non-repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method that allows for a nonrepetitive genomic locus to be labeled using one guide RNA. We construct Casilio dual-color probes to visualize the dynamic interactions of DNA elements in single live cells in the presence or absence of the cohesin subunit RAD21. Using a three-color palette, we track the dynamic 3D locations of multiple reference points along a chromatin loop. Casilio imaging reveals intercellular heterogeneity and interallelic asynchrony in chromatin interaction dynamics, underscoring the importance of studying genome structures in 4D.
Keyphrases
- genome wide
- high resolution
- dna damage
- copy number
- crispr cas
- dna methylation
- gene expression
- transcription factor
- genome editing
- fluorescence imaging
- computed tomography
- small molecule
- single cell
- signaling pathway
- circulating tumor
- photodynamic therapy
- pet imaging
- cell free
- circulating tumor cells
- positron emission tomography