Extended Data Fig. 1: PBE Corrects the -124 C>T Mutation in TERT Promoter.
From: Programmable base editing of mutated TERT promoter inhibits brain tumour growth
a, The DNA region spanning the mutation at chromosome 5, 1,295,113 C>T (-124 C>T) in the TERT promoter locus of the indicated cells was genotyped. Arrows indicate the mutation. b, Diagram of HA–CjABE targeting the -124 C>T mutation under the guidance of a designed sgRNA expressed in an adeno-associated viral vector. HA-tagged CjABE was expressed under the control of the EF-1α core promoter. SgRNA targeting the TERT promoter mutation or a control sgRNA were expressed under the control of the U6 promoter. Both expression cassettes (EF-1α-HA–CjABE and U6-sgRNA) were inserted into an AAV type 2 vector and packaged into virions for cell infection. CjABE, which were expressed by AAVs, bound to the mutated TERT promoter and converted the targeted A base to I via deamination and, subsequently, to C via mismatch repair of tumour cells, leading to correction of the targeted T•A base pair to C•G in the mutant TERT promoter locus, thereby abrogating ETS-driven TERT transcription. c, The indicated cells were infected with AAVs expressing HA–CjABE under the guidance of sgRNAs with or without targeting of the TERT promoter mutation at an MOI of 100 for 72 h. Results of immunoblot analyses using the indicated antibodies are shown. WB, Western blot. The experiment was repeated three times independently with similar results. d–f, Diagram of infection of GBM cells with AAV virus and calculation of MOI is depicted (d). GBM cells were infected by lentivirus expressing puromycin gene (0.01 MOI). After puromycin selection, the expanded and cloned cells were co-infected with AAV viruses expressing CjABE and sgRNA (control sgRNA or 124 sg RNA). CjABE and sgRNA were driven by EF1α and U6 promoter, respectively. qPCR analyses were used to determine the copy numbers of puromycin gene and AAV genes basing on the indicated standard curves (e). MOIs of AAV were determined by the copy numbers of AAV genes vs the copy numbers of puromycin gene (f). The depicted results are the averages from three independent experiments. Values are means ± s.d. g, The indicated cells were infected with AAVs expressing HA–CjABE under the guidance of sgRNAs with or without targeting of the TERT promoter mutation at an MOI of 100 for 72 h. The DNA region spanning the mutation at chromosome 5 (1,295,113 C>T [-124 C>T] in the TERT promoter locus) of the indicated cell lines was genotyped. Arrows indicate the TERT promoter mutation or the WT TERT promoter. h, The time points of the AAV infection and DNA sequencing were shown in upper panel of Fig. 1c. The cells were harvested on day 10 after the first infection with AAVs expressing HA–CjABE under the guidance of sgRNAs with or without targeting of the TERT promoter mutation at an MOI of 100. The DNA region spanning the mutation at chromosome 5 in the TERT promoter locus of the indicated cell lines was genotyped. Arrows indicate the WT TERT promoter. i, The indicated cells were infected with the indicated AAVs expressing HA–CjABE under the guidance of sgRNA with or without targeting of the TERT promoter mutation at an MOI of 100 following the time points showed in upper panel of Fig. 1b. The DNA region spanning the mutation in the TERT promoter locus at chromosome 5 of the indicated cell lines was analysed by Illumina next-generation sequencing at day 10. The number of sequencing reads are presented. Statistical source data are provided in Source Data Extended Data Fig. 1.
