Elucidation of the molecular mechanism of the breakage-fusion-bridge (BFB) cycle using a CRISPR-dCas9 cellular model

Author:

Singh Manrose1,Raseley Kaitlin2,Perez Alexis M1,MacKenzie Danny1,Kosiyatrakul Settapong T3,Desai Sanket1,Batista Noelle1,Guru Navjot1,Loomba Katherine K1,Abid Heba Z2ORCID,Wang Yilin2,Udo-Bellner Lars1,Stout Randy F1,Schildkraut Carl L3,Xiao Ming24,Zhang Dong15ORCID

Affiliation:

1. Department of Biomedical Sciences, College of Osteopathic Medicine, New York Institute of Technology , Old Westbury, NY  11568 , USA

2. School of Biomedical Engineering, Science and Health System, Drexel University , Philadelphia , PA 19104 , USA

3. Department of Cell Biology, Albert Einstein College of Medicine , Bronx , NY  10461 , USA

4. Center for Genomic Sciences and Center for Advanced Microbial Processing, Institute of Molecular Medicine and Infectious Disease, Drexel University College of Medicine , Philadelphia , PA, 19102 , USA

5. Center for Cancer Research, New York Institute of Technology , Old Westbury, NY  11568 , USA

Abstract

Abstract Chromosome instability (CIN) is frequently observed in many tumors. The breakage-fusion-bridge (BFB) cycle has been proposed to be one of the main drivers of CIN during tumorigenesis and tumor evolution. However, the detailed mechanism for the individual steps of the BFB cycle warrants further investigation. Here, we demonstrate that a nuclease-dead Cas9 (dCas9) coupled with a telomere-specific single-guide RNA (sgTelo) can be used to model the BFB cycle. First, we show that targeting dCas9 to telomeres using sgTelo impedes DNA replication at telomeres and induces a pronounced increase of replication stress and DNA damage. Using Single-Molecule Telomere Assay via Optical Mapping (SMTA-OM), we investigate the genome-wide features of telomeres in the dCas9/sgTelo cells and observe a dramatic increase of chromosome end fusions, including fusion/ITS+ and fusion/ITS−. Consistently, we also observe an increase in the formation of dicentric chromosomes, anaphase bridges, and intercellular telomeric chromosome bridges (ITCBs). Utilizing the dCas9/sgTelo system, we uncover many interesting molecular and structural features of the ITCB and demonstrate that multiple DNA repair pathways are implicated in the formation of ITCBs. Our studies shed new light on the molecular mechanisms of the BFB cycle, which will advance our understanding of tumorigenesis, tumor evolution, and drug resistance.

Funder

New York Institute of Technology

Publisher

Oxford University Press (OUP)

Reference62 articles.

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