Fig. 2

CRISPR/Cas9-mediated gene rearrangement induced endogenous RELA^FUS1 in human cultured cells. a Experimental strategy for the CRISPR/Cas9-mediated gene rearrangement to induce endogenous RELA^FUS1 in 293T cells. Blue and red boxes represent exons of the C11orf95 and RELA genes, respectively. Black arrowheads indicate cleavage sites by the sgRNAs. Red arrows indicate PCR primer pairs for the fusion-junction detection. b sgRNA combination for the CRISPR/Cas9-mediated gene rearrangement in 293T cells. c RT-PCR detection of RELA^FUS1 transcript in 293T cells. RNA was extracted from 293T cells that were transiently transfected with the sgRNA combination-1, 2 or mock vectors (left panel) The RNA was then subjected to RT-PCR analyses for C11orf95-RELA fusion junction (left-top) and intact RELA detection (left-bottom panel). The electropherograms of the Set-1 and 2 PCR products are shown in right-top and right-bottom panels, respectively. NT, non-treated parental 293T cells. d Western blot analysis for RELA^FUS1 detection in the gene-edited 293T cells. The set-1 and set-2 sgRNAs were transiently transfected into 293T cells. Cell lysates were subjected to immunoblot blot analysis with the indicated antibodies. Upper (red) and lower (black asterisk) bands in the RELA antibody detection indicate RELA^FUS1 and endogenous RELA protein, respectively. Cell lysate extracted from pTomo-RELA^FUS1-induced brain tumor tissue serves as a positive control for RELA^FUS1 protein expression