Fig. 1: Design of optogenetic switches for genomic cell engineering.
A–C Modes of function for the optogenetic gene switches. Light exposure activates the expression of a gene of interest (GOI) via recruitment of the transactivation domain VP16 to a minimal CMV promoter (Pmin). A RED gene switches utilize the PhyBN/PIF6APB interaction and either E or TetR as the DNA-binding domain specific for the etr8 or TCE DNA elements, respectively. Red light (R) activates PhyBN and initiates binding to PIF6APB, and thus recruitment of the reconstituted TF to the target sequence of the respective DNA-binding domain to the synthetic promoter, whereas far-red light causes dissociation. B BLUE gene switch implemented either on one (SINGLE) or two (DUAL) independent vectors. Blue light causes the binding of LOVpep to ePDZb via relaxation of helix Jα exposing the otherwise caged ePDZb-interacting peptide. C The EL222 single-component gene switch exhibits natural photo-regulated DNA binding capacity to the C120 operator sequence in response to blue light. Fusion of EL222 to VP16 results in a functional optogenetic TF for eukaryotic cells. D General architecture of transposition-competent vectors encoding photoswitches. E Architecture of attenuated (Ra, δ spacing, no ITRs), transposition-incompetent (R, no ITRs), and transposition-competent (Rt) reporter constructs utilizing SEAP as the GOI (see Table S1, Supplementary Information, for details). TCE contains seven tetO repeats. F Functional tests of tTA (TetR-VP16) and eTA (E-VP16)-activatable SEAP reporter constructs as in (E) using transient transfection together with the respective activators (Act) into CHO-K1 cells. p-values: tetO7-R, 1.622e−4; tetO7-Rt, 1.319e−4; etr8-R, 8.976e−5; etr8-Rt, 2.577e−3. G Transient transfection test of transposition-competent vectors for the REDTET and REDE system as in (D) for E and TetR-specific systems in CHO-K1 cells. The attenuated TetR-specific reporter construct contained 13 tetO operator repeats. p-values: tetO13-Ra, 2.332e-7; tetO7-R, 2.331e-7; tetO7-R, 6.052e-8; etr8-Ra, 1.203e-5; etr8-R, 7.520e-6; etr8-Rt, 1.922e-5. H Transient test of BLUESINGLE and BLUEDUAL transposition-competent vectors in CHO-K1 cells as in (D). p-values: Single-Ra, 1.514e-6; Single-R, 6.492e-8; Single-Rt, 4.344e-6; Dual-Ra, 1.027e-3; Dual-R, 6.120e-7; Dual-Rt, 3.229e-5. I Transient test of the transposition-competent vector encoding the EL222 photoswitch in CHO-K1 cells. p-value: 8.224e-7. F–I Used reporter constructs as in (E). Data represent mean values with one standard deviation of four biological replicates compared with two-sided independent Student’s t-tests. **, 1e-3 <p ≤ 1e-2; ***, 1e-4 <p ≤ 1e-3; ****, p ≤ 1e-4. Source data are provided with this paper.
