Fig. 1: Design and optimization of the far-red light-induced split Cre-loxP system (FISC system).

a Schematic representation of the FISC system. Cre recombinase was split into two fragments: CreN59 (residues 1–59) fused to Coh2 driven by a constitutive promoter (P_hCMV) and CreC60 (residues 60–343) fused to DocS driven by the far-red light (FRL, 730 nm)-inducible promoter (P_FRLx). Upon FRL illumination, the photoreceptor BphS is activated to convert intracellular guanylate triphosphate (GTP) into cyclic diguanylate monophosphate (c-di-GMP). The cytosolic c-di-GMP production induces binding of the far-red light-dependent transactivator FRTA (p65-VP64-BldD) to its synthetic promoter P_FRLx to drive DocS-CreC60 expression. Consequently, the catalytic activities of Cre recombinase can be restored once the two Cre fragments assemble based on affinity interactions of their respective Coh2 and DocS fusion domains, enabling to excise DNA sequences flanked by loxP sites. b Schematic depicting the genetic configuration of constructs used in the FISC system. pA, polyadenylation signals; YhjH, the bacterial c-di-GMP phosphodiesterase; DocS, dockerin S from C. thermocellum complexed with Coh2; L_1-11, different linkers from 1 to 11 (Supplementary Table 1), Coh2, an anchoring protein from C. thermocellum; loxP, the specific Cre recombinase binding site; STOP, a terminator containing pA to prevent transcription; GOI, gene of interest. c Schematic depicting different genetic configurations of the FRL-inducible promoters P_FRLx. 3*whiG, three copies of BldD-specific binding sequence; P_hCMVmin, minimal version of P_hCMV; P_min, minimal eukaryotic promoter; TATA, minimal eukaryotic promoter with only TATA box. d Schematic depicting the time schedule for the FISC experimental procedure with mammalian cells. e Optimization of the different transfection amounts for CreN59-Coh2 expression and light-inducible P_FRLd-driven DocS-CreC60 expression. HEK-293 (6 × 10⁴) cells were cotransfected with pXY137 (P_hCMV-p65-VP64-BldD-pA::P_hCMV-BphS-P2A-YhjH-pA, 100 ng), pGY125 (P_hCMV-loxP-STOP-loxP-SEAP-pA, 200 ng), pXY110 (P_hCMV-CreN59-L0-Coh2-NES-pA) and pXY133 (P_FRLd-NLS-DocS-L0-CreC60-pA) from 5 to 100 ng at a ratio of 1:1 (w/w), and then illuminated for 6 h with FRL (1.5 mW cm^-2, 730 nm) once each day for 2 days. SEAP expression in the culture supernatants was profiled at 48 h after the first illumination (n = 3 independent experiments). f Optimization of the different linkers (L1–L11) between the CreN59 and Coh2 domains, as well as the CreC60 and DocS domains. The 6 × 10⁴ HEK-293 cells per well were cotransfected with pXY137 (100 ng), pGY125 (200 ng), and Docs-CreC60 and Coh2-CreN59 with different combinations of the linkers (10 ng/10 ng) (Supplementary Table 2); these were illuminated as described in e, followed by SEAP expression in the culture supernatants profiled at 48 h after the first illumination (n = 3 independent experiments). The orange frame in (e, f) marks the best-performing condition. e, f Data represent the mean ± SD. Source data for this figure are available in the Source data file.