Poster abstracts
Poster number 36 submitted by Dilshodbek Nishonov
CRISPR-CLIP: A novel tool for stabilizing artificial chromatin loops in live-cells
Yu-Chieh Chung (The Ohio State University), Siou-Luan He (The Ohio State University), Dilshodbek Nishonov (The Ohio State University), Nevin Wise (The Ohio State University)
Abstract:
Chromatin looping is an essential process for proper genome organization and precise control of transcriptional regulation. However, the mechanisms by which chromatin interactions influence transcription remain under active investigation. Recent advances in CRISPR technology have enabled powerful new tools for studying the causal links between chromatin loops and transcription, including systems that induce artificial chromatin loops by dimerizing two DNA-bound CRISPR/dCas9/guide RNA (gRNA) complexes. While these systems can successfully manipulate chromatin folding, their efficiency and capacity to induce long-range chromatin interactions remain limited. To overcome these limitations, we redesigned our previously developed CRISPR-Sirius system, originally created for live-cell imaging of specific genomic loci, into CRISPR-CLIP, a novel tool to physically manipulate chromatin at a kilobase- and megabase-scale distances. In CRISPR-CLIP, a dual-function gRNA was formed by fusing two CRISPR-Sirius gRNAs into a single CLIP gRNA, which, upon binding to DNA, forces two genomic loci into stable close proximity. We verified CRISPR-CLIP in live human cells by targeting pairs of distal loci with varying genomic distances and visualizing their colocalization using fluorescently labeled MS2 and PP7 RNA hairpins engineered into the CLIP-gRNA scaffold. Quantitative measurements confirmed successful and sustained chromatin looping. In addition to stabilizing individual artificial loops, CRISPR-CLIP was also used to contract a large chromosomal domain (~17 Mb) on the long arm of chromosome 19. To examine the effects of chromosome contraction on gene expression, we conducted RT-PCR following CRISPR-CLIP-induced contraction. Preliminary results suggest that total mRNA levels were not significantly influenced, indicating that reducing chromosome volume alone is insufficient to repress transcription. Overall, CRISPR-CLIP represents a novel live-cell tool for dissecting the role of chromatin architecture in transcriptional regulation. By enabling precise, synthetic looping of cis-regulatory elements, it opens new avenues for exploring gene regulation and developing future clinical therapeutics for genetic disorders.
References:
Ma, H., Tu, LC., Naseri, A. et al. CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat Methods 15, 928–931 (2018). https://doi.org/10.1038/s41592-018-0174-0
Keywords: RNA, CRISPR, chromatin