Fig. 4. Identification and characterization of large PE3-mediated insertions in single cell-derived clones.

a Schematic representation of the large insertions observed at MTOR exon 44 from single cell-derived K562 clones harboring the MTOR-I2017T hyperactivating mutation. K562 cells stably expressing the mSc-TOSI reporter were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and MTOR. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA was harvested after co-selection. TOPO cloning and Sanger sequencing were performed to characterize the large insertions. The pegRNA and its PAM sequence are represented in orange and dark orange, respectively. The nick sgRNA and its PAM sequence are represented in blue and dark blue, respectively. b Same as in a for large insertions at MTOR exon 53 from single cell-derived K562 clones harboring the MTOR-E2419K hyperactivating mutation.