CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

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 low signal-to-noise ratios (SNRs), and are thus mostly applicable to repetitive sequences. Here, we report a versatile CRISPR/Casilio-based 20 imaging method, with an enhanced SNR, that allows for one nonrepetitive genomic locus to be labeled using a single sgRNA. We constructed Casilio dual-color probes to visualize the dynamic interactions of cohesin-bound elements in single live cells. By forming a binary sequence of multiple Casilio probes (PISCES) across a continuous stretch of DNA, we track the dynamic 3D folding of a 74kb genomic region over time. This method offers unprecedented resolution and 25 scalability for delineating the dynamic 4D nucleome.One Sentence Summary: Casilio enables multiplexed live cell imaging of nonrepetitive DNA loci for illuminating the real-time dynamics of genome structures.

FISH Going Meso-Scale: A Microscopic Search for Chromatin Domains

“…CRISPR/dCas9-based technology evolves exceedingly fast in terms of adapted labeling strategies to enhance signal-to-noise ratio and single locus visibility within cell nucleus ( Wu et al, 2019 ). For instance, multicolor and high-resolution live cell tracking of loci and monitoring of inter-loci distances was achieved using either three dCas9 with different sgRNA binding specificity ( Ma et al, 2015 ) or by engineering sgRNA to harbor RNA aptamers, recognized by cognate binding proteins ( Clow et al, 2020 ).…”

Live cell imaging of DNA and RNA with fluorescent signal amplification and background reduction techniques

“…A CRISPR/Casilio-based imaging method was also developed recently for labeling non-repetitive genomic locus with only one sgRNA. The scaffold of the sgRNA contains 15 copies of PUF binding sites c (15× PBSc) (Clow et al, 2022). In the system, each sgRNA can recruit 15 fluorescent proteins, and the labeling specificity was evaluated by co-labeling with FISH probe.…”

“…A ~500 kb loop domain between the IER5L promoter (IER5L-P) and its super-enhancer (IER5L-SE) was observed to be lost upon the depletion of RAD21 in a previous study (Rao et al, 2017). In an engineered HCT116 cell line with endogenous RAD21 fused to an auxin-inducible degron, the IER5L-P and IER5L-SE loci were labeled using a CRISPR/Casilio-based system (Clow et al, 2022). When treated with auxin, the spatial distance between the two loci significantly increased compared to the untreated control, demonstrating the involvement of RAD21 in loop domain formation and intrachromosomal interaction.…”

Women’s contribution in understanding how topoisomerases, supercoiling, and transcription control genome organization

“…For high-resolution distance mapping, Mateo et al (2019) developed ORCA (Optical Reconstruction of Chromatin Architecture), an optical method that allows tracing of DNA in 2 kb steps in single cells. To study genome dynamics with high spatiotemporal resolution, Clow et al (2022) optimized the Casilio (CRISPR-Cas9-Pumilio) system (Cheng et al, 2016) for the simultaneous imaging of multiple non-repetitive DNA sequences in live cells, exploiting defective Cas9 and engineered sgRNAs recognized by fluorescently-tagged proteins. To probe proximity at specific genomic loci, Mota et al (2022) recently developed FRET-FISH (Fluorescence Resonance Energy Transfer combined with DNA Fluorescence In Situ Hybridization), a method that could be applied in the future to study the formation of loops and condensates in single cells.…”