TMEPAI genome editing in triple negative breast cancer cells
Abstract: 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.
Keywords: CRISPR/Cas9; gene
editing; knock-out cell lines
Author: Bantari W.K. Wardhani,
Meidi U. Puteri, Yukihide Watanabe, Melva Louisa, Rianto Setiabudy, Mitsuyasu
Kato
Journal Code: jpkedokterangg170229
