CRISPR/Cas9-system mediated deletion of the Neurofibromin 1 tumour suppressor gene in human gliomas

Abstract

Conventional therapies to treat glioblastoma multiforme (GBM) are unable to completely eliminate tumour cells and rapid emergence of post-therapeutic tumour lesions results in high patient mortality rates. Clinical observations have suggested the presence of a proneural to mesenchymal transition (PMT) of glioma cells at the time of tumour recurrence. The Neurofibromin 1 (NF1) tumour suppressor gene is frequently lost or mutated in gliomas of the mesenchymal subtype. However little is known of the molecular mechanisms underlying NF1 inactivation, and its potential role in mediating PMT has yet to be studied. This study aims to utilize and develop the CRISPR/Cas9 gene editing system as a molecular tool to investigate the role of NF1 in brain tumour progression. Construction of sgRNAs, assembly into PX459 CRISPR plasmids and transfection of the plasmids into HEK293T and U87 cell lines were successful, revealing a robust transfection efficiency of 90%. Preliminary results showed a 10 to 60% reduction of the NF1 protein in CRISPR-transfected HEK293T and U87 cells. Quantitative PCR of pooled puromycin-selected HEK293T-transfected cells demonstrated a 94% downregulation of NF1 mRNA transcripts. With further optimization of the CRISPR/Cas9 system, molecular mechanisms that mediate PMT in glioma cells can be fully elucidated.