Abstract: In order to study the function of growth differentiation factor NODAL（nodal growth differentiation factor）,the cell line with NODAL gene knockout needs to be established. Two different CRISPR/Cas9 expression vectors targeting different sites and one targeting vector for homologous recombination were constructed and then transfected into triple negative breast cancer cell line MDA-MB-231 by electrotransfection. The genotypes after purinomycin resistance screening and single-cell clones selection were identified by PCR and sequencing,and protein expression was detected by Western blot. Eight single-cell clones identified as positive by allelic homologous recombination assay were re-identified by non-homologous recombination and large fragment deletion analyses. Only one single-cell clone had a frame-shift mutation in the target site. A large fragment deletion occurred at the target sites of the 4 single-cell clones,and the wild-type alleles were detected at the target sites of the 4 single-cell clones. Western blot showed that NODAL expressions in only 2 single-cell clones were not detected,and NODAL expressions were decreased in 3 single-cell clones,and no significant changes in NODAL expressions were found in other 3 single-cell clones. Thus,the single-cell clones with NODAL gene knockout are obtained based on genotype identification and Western blot,which lays a foundation for further research on the molecular mechanism of NODAL in breast cancer cells.

Key words: CRISPR/Cas9, breast cancer, gene knockout, NODAL gene