Fig. 1: Strategy and in vitro CRISPR-mediated disruption of EWSR1-FLI1.

a Scheme representing type 1 and 2 EWSR1-FLI1 FOs, illustrating the genomic structure with exon arrangement and sites of fusion. sgRNAs targeting introns 3 and 6 of EWSR1 and 6 and 8 of FLI1 are indicated. b Schematic representation of the all-in-one lentiviral vector for simultaneous expression of two sgRNAs, Cas9 and eGFP regulated by the U6, H1, and EFS promoters. c, Genomic PCR analysis of edited and control A673 cells using oligonucleotides flanking the DNA targeted by sgE3 and sgF8 (n = 3, independent studies). The 300 bp PCR product denotes deletion of the DNA fragment between the loci targeted by sgE3 and sgF8. PCR analysis was performed on extracted DNA of cells at day 2, 4, and 6 post-transduction (pt). ALBUMIN was used as an internal control of the PCR reaction. Bottom panel shows a representative Sanger sequencing chromatogram of the PCR products. d RT-PCR products from edited and control A673 Ewing sarcoma cells (n = 3, independent studies). Analysis was done using extracted RNA of cells at day 2, 4, and 6 pt. Arrows depict the sizes of wild-type (961 bp) and deleted (150 bp) RT-PCR products. GAPDH was used as an internal control of the RT-PCR reaction. Bottom panel shows a representative Sanger sequencing chromatogram of RT-PCR products. e Western blotting of EWSR1-FLI1 in A673 cells. Analysis was done using total protein extracted from cells at day 3, 6, and 10 post-transfection using an antibody specific for FLI1. GAPDH was used as an internal control of the assay. LTR: Long term repeat; P2A: porcine teschovirus-1 2A self-cleaving peptide; WPRE: Virus (WHP) posttranscriptional regulatory elements.