An old teddy bear with an eye missing, a dog-eared children’s book, or a ragged musty-smelling blanket; we all “cling” on to certain treasured objects, although we usually wish to keep them secret from the world! However, this isn’t the case for researchers from the laboratory of John Rinn (Harvard University, Cambridge, USA), who have employed an exciting new CRISPR-based approach to show all and sundry how specific regions on different chromosomes CLING to each other over time and coordinate gene regulation.
In this context, CLING stands for CRISPR live-cell imaging, and the talented team applied this novel technique to study the dynamic genome contacts made between non-homologous chromosomes over time. As an example, non-homologous contacts occur between chromosome A and chromosome B (interchromosomal), while homologous contacts arise between sister chromatids of chromosome A (intrachromosomal). While highly useful, current techniques employed to assess genome organization, including conventional imaging-based techniques and genome-wide chromosome conformation capture (Hi-C), present drawbacks that do not permit the assessment of locus-specific chromosomal contacts over time.
So how does CLING work? Simply put, the technique employs dCas9 and two pools of single-guide RNAs (sgRNAs); one sgRNA pool targets one chromosomal locus and contains a specific RNA-aptamer motif fused to a fluorescent label, while another sgRNA pool targets the interacting chromosomal locus and contains the required RNA-binding protein fused to a different fluorescent label. When applied to a single mammalian cell, the group simply recorded the movements of the fluorescent-labelled regions to describe the dynamics and the frequency of non-homologous chromosomal contacts (NHCCs).
What did the authors discover with this clingy new CRISPR-based technique?
- CLING and Hi-C techniques readily detect similar homologous chromosomal contacts (HCCs)
- Examples include interactions between the FIRRE locus and the DXZ4 macrosatellite repeat or the inactive-X CTCF-binding contact element (ICCE)
- Interestingly, NHCCs occur at a similar frequency to HCCs and display similar levels of stability
- NHCC examples include interactions of the FIRRE locus with YPEL4 or ATF4 loci or the interaction of the CISTR-ACT locus with the SOX9 locus
- NHCCs occur in the majority of the mouse and human cells assessed and exhibit three-dimensional conservation
- However, NHCCs occur over more considerable distances compared to HCCs and, therefore, may be missed by Hi-C analysis
- The group suggest that this proves the advantage of live-cell imaging in assessing chromosomal contacts
The authors hope that the spatiotemporal dimension of live-cell imaging afforded by CLING will help to develop our understanding of how both short distance HCCs and long distance NHCCs control gene expression.
So don’t get too attached to old methodologies when it comes to chromatin contacts; instead CLING on to something new at Molecular Cell, March 2018.