Poster abstracts
Poster number 3 submitted by Nevin Wise
Large-scale chromosome silencing in live cells by CRISPR and engineered guide RNAs
Yu-Chieh Chung (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:
Aneuploidy and oncogenic extrachromosomal DNA are cancer hallmarks, speeding up cancer evolution and correlating with metastasis. Clustered, regularly interspaced short palindromic repeats (CRISPR) have been repurposed to edit individual genes in human cells with high precision. However, tools for silencing multiple genes remain in need. In this research, we developed a new nanodevice, CRISPR-CLIP, for simultaneous engineering and visualizing the human genome in live cells. Built on our previously developed CRISPR-based imaging tool, CRISPR-Sirius, for tracking chromatin movement in real-time, we genetically fused two CRISPR-Sirius gRNAs, named CLIP-gRNA, and delivered them to U2OS cells. One CLIP-gRNA successfully brought the two loci (A03 and IDR3) that are ~16 Mb into proximity, demonstrated by the significantly reduced loci distance. To condense large chromosomal domains, we utilized repetitive sequences in the human genome. We successfully condensed a gene-rich chromosome, chromosome 19 (Chr19), at its long arm using a CLIP-gRNA targeting ~836 times within the 17Mb region. Significant volume reduction was observed on the CRISPR multiple CLIPs (CRISPR-mCLIP) cell compared to the control (two separated gRNA). Overall, the use of CRISPR and gRNA as a chromosomal silencing mechanism suggests possible therapeutic approaches to treat aneuploidy and oncogenic extrachromosomal DNA.
References:
Kim, H. et al. Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers. Nat. Genet. 52, 891–897 (2020).
Akdemir, K. C. et al. Disruption of chromatin folding domains by somatic genomic rearrangements in human cancer. Nat. Genet. 52, 294–305 (2020).
Jiang, F., Zhou, K., Ma, L., Gressel, S. & Doudna, J. A. A Cas9–guide RNA complex preorganized for target DNA recognition. Science 348, 1477–1481 (2015).
Kim, J. H. et al. LADL: light-activated dynamic looping for endogenous gene expression control. Nat. Methods 16, 633–639 (2019).
Ma, H. et al. CRISPR-Sirius : RNA scaffolds for signal amplification in genome imaging. Nat. Methods 15, (2018).
Keywords: Gene Silencing , Live-Cell Imaging , CRISPR