Extended Data Fig. 4: Generation and characterization of CymR clonal cell lines.
From: An engineered UGA suppressor tRNA gene for disease-agnostic AAV delivery
(a) Schematics showing the gene editing strategy to integrate the CymR gene into the AAVS1 locus in HEK293 cells. SgRNA: single guide RNA. LHA: left homology arm. SA: splicing acceptor. P2A: Porcine teschovirus-1 2 A peptide. T2A: Thosea asigna virus 2A peptide. PuroR: puromycin resistance gene. bGHpA: bovine growth hormone polyadenylation signal. RHA: right homology arm. (b) Cartoon showing the procedure to generate the CymR stable cell pool. (c) Cartoon showing the mechanism of CuO/CymR-controlled EGFP reporter. In the absence of cumate (left), CuO-CymR binding blocks EGFP expression. In the presence of cumate (right), it binds to CymR, preventing the inhibitory effect on EGFP expression. (d) EGFP fluorescence images from HEK293 or various clonal cell lines transfected with the CuO/CymR-controlled EGFP reporter plasmids in the absence or presence of cumate at 30 µg/mL. The screening was performed once. (e) Tabulation of large-scale production yield of rAAV9.2xCuO-hNFS-R8 with CymR clonal cell lines. Vg: vector genome.
