CRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci with one guide RNA per locus.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 35387989.
- Also identified by DOI 10.1038/s41467-022-29343-z and PMC identifier 8987088.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
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.
Medical subject headings
- Clustered Regularly Interspaced Short Palindromic Repeats
- RNA, Guide, CRISPR-Cas Systems