Bowel cancer genetics: investigating chromosomal instability by CRISPR knockout panels


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Fadipe Adetokunbo1,Lennard YW Lee2
1Merton College, Oxford,2Institute of Cancer Sciences, University of Birmingham

Abstract

Background

Chromosomal instability (CIN) is an important, but poorly understood molecular feature of colorectal cancer (CRC). The underlying genetic causes are not fully understood and an area of interest as patients with CIN have a poorer prognosis.

Method

The mitocheck database was interrogated to identify genes linked to CIN. This was then cross-linked to The Cancer Genome Atlas to identify 31 putative gene targets linked to CRC. Genes identified include those previously implicated in CRC carcinogenesis, such as FGFR-2, RGMA and DOCK-2. The Diploid HCT 116 cell lines was used as a model cellular system and CRISPR/Cas9 sgRNA targetting these 31 genes were generated (2 for each gene). These constructs were then individually transduced in colonies of HCT116. Colonies were then stained with propidium iodide and doublet discrimination flow cytometry was performed to determine degree of induction of CIN.

Results

There was a strong inverse correlation between the aneuploidy and diploidy shown in the samples (r=-0.93, p= <0.00001). Of the 31 genes, two loci were be flagged as potentially of interest, NUP214-2 and MYH9-2. Knock out of these genes demonstrated a trend to increasing levels of CIN.

Conclusion

This study determined that CRISPR/Cas9 knockout models is a feasible approach to allow high throughput screening of genes associated with CIN. Further analyses of NUP214-2 and MYH9-2 in causing CIN is warranted.