Fig. 3
CRISPR/Cas9-mediated gene rearrangement generated induces oncogenic 2700081O15Rik-Rela fusion in mice. a Experimental strategy for the CRISPR-Cas9-mediated gene rearrangement to induce mRelafus. Orange and purple boxes represent exons of Rela and 2700081O15Rik gene, respectively. Black arrowheads and Red arrows indicate cleavage sites by the sgRNAs and PCR primer pairs for the fusion-junction detection, respectively. b sgRNA combinations for the CRISPR/Cas9-mediated gene rearrangement in NIH3T3 cells. c RT-PCR detection of the mRelafus transcript in the NIH3T3 cells. RNA was extracted from NIH3T3 cells transiently transfected with the sgRNA combination-1, 2 or mock vectors (left panel) and then subjected to RT-PCR analyses for 2700081O15Rik-Rela fusion junction (left-top) and intact Rela detection (left-bottom panel). The electropherogram of the PCR products (Set-1 and 2) is shown in the middle and right panel. NT, non-treated parental NIH3T3 cells. d Schematic presentation of the pTomo-LV-EDIT-mRelafus vector construct for simultaneous multiple sgRNA delivery into the mouse brain. mU6, murine U6 promoter. See also Additional file 6: Fig. S1A. e and f Kaplan–Meier survival curves and Summary of the LV-EDIT-mRelafus vector injections. Lentiviruses were injected into neonatal pups brain in Nestin-Cre+/−;Cag-Cas9+/+ mice (n.s., not significant. M, medium titer; H, higher titer; n/a, not applicable). g and h Representative H&E and IHC stains for RELA, GFAP and GFP antibodies of the endogenous mouse Rela fusion-induced brain tumor in Nestin-Cre+/−;Cag-Cas9+/+ mice (n = 3). i RT-PCR detection of mRelafus transcript in the LV-EDIT-mRelafus-induced brain tumor and electropherograms of the PCR products. Sanger sequencing analyses of the PCR products identified two types of in-frame fusion transcripts corresponding to human RELAFUS1 (middle panel) and RELAFUS4 (right panel) in this tumor (NBT-115). See also Additional file 8: Fig. S3C. j Western blot analysis in the mouse tumor-derived cell line (NBT-514) as shown in Additional file 8: Fig. S3C. Upper and lower bands in the RELA antibody detection indicate mRelafus1 and endogenous Rela protein, respectively