CRISPR-Based Strategy for imaging Extrachromosomal DNA in live tumour cells

Chitra Roy, University of Calcutta

In 1965, a group of British oncologists viewed DNA fragments which were floating around chromosomes in cancer cells. For decades, there has been largely ignored as cellular debris. Recent advancements of technology in the field of genetics and cellular biology has paved a way for better understanding of complex diseases like cancer and revealed that those DNA fragments, now known as Extrachromosomal DNA (ecDNA), play a significant role in promoting cancer growth and in the development of tumour resistance. 

ecDNA is distinct circular DNA entities which contains functional genes and are located outside cells’ chromosome. On August 17, 2020, a group of researchers at the University of California, San Diego, reported that ecDNA is very commonly found in a minimum of 14% of human tumours. These researchers, for the first time, gave direct evidence that multiplication of this circular ecDNA (now known as ecDNA Oncogene Amplification) occurs in a wide range of tumour types. They also observed that ecDNA amplification is a special event in cancer and does not occur in normal tissue.

ecDNA has this tremendous capacity to make huge copies of themselves by replicating within the cancer cells. This results in an increased copy of oncogenes which can be passed down to the next generation in an asymmetric manner. This asymmetry arises due to an important feature of this ecDNA, that is, it lacks centromeres, which is normally present in the centre of chromosomes and is essential for distributing the DNA evenly between daughter cells. But for ecDNA, it is divided randomly and there are unequal distribution and copy number variation among cancer cell subpopulation.

However, this is just a hypothesis which lacks sufficient evidence. Moreover, the presence of ecDNA causes intratumor heterogeneity which creates a barrier to treat cancer. Recently, a team of researchers came up with new approaches to address these scientific problems. They showed using fluorescence in-situ hybridization (FISH) that single-cell ecDNA copy number in tumour cells in vitro and four glioblastomas tumour tissue sample followed a Gaussian distribution, giving direct evidence that ecDNA is segregated unevenly during the event of mitosis and has high copy number variation. 

To understand how ecDNA promotes tumour heterogeneity, they established a CRISPR-based approach to study it in live cells. The researchers exploited the mechanism of formation of ecDNA for devising their study. ecDNA is formed by DNA breakage following end-to-end ligation which results in one or more breakpoint junctions and the sequences of these junctions are unique. This presents an opportunity in designing single guide-RNA (sgRNA) to either tag or target ecDNA. The researches employed a hybrid technique where they used dead Cas9 and RNA-binding to recruit multiple fluorescent molecules to specific sgRNA target locus. 

This study gives a whole new dimension in expanding our understanding of the potential of ecDNA in tumour and also, how revolutionizing gene-editing tools like CRISPR-Cas9 can enable us to engineer a live-cell ecDNA tracking system! 

Also read: DO COVID-19 PATIENTS BENEFIT FROM TOCILIZUMAB AT ALL?

Reference: https://www.biorxiv.org/content/10.1101/2020.10.20.335216v1

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