Poster abstracts
Poster number 110 submitted by Dilshodbek Nishonov
Live-Cell Chromatin Looping by CRISPR and Engineered gRNAs
Yu-Chieh Chung (Department of Biological Chemistry and Pharmacology, The Ohio State University), Siou-Luan He (Department of Biological Chemistry and Pharmacology, The Ohio State University), Dilshodbek Nishonov (Department of Biological Chemistry and Pharmacology, The Ohio State University), Nevin Wise (Department of Biological Chemistry and Pharmacology, The Ohio State University), Li-Chun Tu (Department of Biological Chemistry and Pharmacology, The Ohio State University)
Abstract:
Chromatin looping is critical in organizing the genome to allow for controlled regulation of transcriptional activity. However, the extent to which chromatin-chromatin interactions causally affect gene expression is unclear. Forced chromatin looping methods using CRISPR offer an additional avenue to study cause-effect relationships between chromatin state and transcription. Although there are existing CRISPR-based chromatin looping systems, limitations exist in their capability to form long-range looping interactions and in their efficiency. Here, we repurposed our existing CRISPR-Sirius system used for live cell imaging into CRISPR-CLIP, a novel method to physically manipulate chromatin folding at a kilobase and megabase scale. The transformation was achieved by fusing two CRISPR-Sirius guide RNAs (gRNAs) into a single CLIP gRNA to force loci pairs to localize into proximity. We successfully verified our system by targeting loci pairs within individual human chromosomes with varying genomic distances and by visualizing the colocalization of fluorescent signals from the MS2 and PP7 hairpins engineered in the CLIP gRNA scaffold. The colocalization was confirmed by measuring the physical distances between the loci pairs. Furthermore, we achieved large-scale chromosome contraction (~17 Mb) by targeting multiple CLIP gRNAs to the repetitive sequence within the chromosome 19 q arm. RT-PCR was performed to measure the changes in gene expression after the chromosome contraction. We did not observe significant changes in expression in response to the volume reduction, suggesting that chromatin volume reduction alone is not sufficient to repress gene expression. Overall, CRISPR-CLIP is a live-cell tool that can serve to answer questions about the role of chromatin organization at different genomic scales in gene expression and help elucidate how long-range interactions between cis-regulatory elements function.
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: gRNA, chromatin, CRISPR