TMEPAI genome editing in triple negative breast cancer cells

• Main Authors: Bantari W.K. Wardhani, Meidi U. Puteri, Yukihide Watanabe, Melva Louisa, Rianto Setiabudy, Mitsuyasu Kato
• Format: Article
• Language: English
• Published: Faculty of Medicine Universitas Indonesia 2017-05-01
• Series: Medical Journal of Indonesia
• Subjects: CRISPR/Cas9; gene editing; knock-out cell lines
• Online Access: http://mji.ui.ac.id/journal/index.php/mji/article/view/1871

Background: Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) is a powerful genome editing technique. It consists of RNA-guided DNA endonuclease Cas9 and single guide RNA (gRNA). By combining their expressions, high efficiency cleavage of the target gene can be achieved, leading to the formation of DNA double-strand break (DSB) at the genomic locus of interest which will be repaired via NHEJ (non-homologous end joining) or HDR (homology-directed repair) and mediate DNA alteration. We aimed to apply the CRISPR/Cas9 technique to knock-out the transmembrane prostate androgen-induced protein (TMEPAI) gene in the triple negative breast cancer cell line. Methods: Designed gRNA which targets the TMEPAI gene was synthesized, annealed, and cloned into gRNA expression vector. It was co-transfected into the TNBC cell line using polyethylenimine (PEI) together with Cas9-GFP and puromycin resistant gene vector. At 24-hours post-transfection, cells were selected by puromycin for 3 days before they were cloned. Selected knock-out clones were subsequently checked on their protein levels by western blotting. Results: CRISPR/Cas9, a genome engineering technique successfully knocked-out TMEPAI in the Hs578T TNBC cell line. Sequencing shows a frameshift mutation in TMEPAI. Western blot shows the absence of TMEPAI band on Hs578T KO cells. Conclusion: TMEPAI gene was deleted in the TNBC cell line using the genomic editing technique CRISPR/Cas9. The deletion was confirmed by genome and protein analysis.