Fig. 1: Design of PhoBIT1 as a light-OFF switch and its application to control gene expression.

a Schematic of a mitochondria translocation assay to assess light-dependent dissociation between mitochondria-anchored ssrA and engineered photo-responsive sspB. LOV2 is inserted into sspB to generate a hybrid protein, sspB(LOV2). b, c 2D topology (b) and 3D structure (c) of sspB (green; PDB entry: 1TWB) with LOV2 insertion sites indicated by spheres. The best-performing insertion site, S5, was highlighted in magenta. d Quantification of light-induced mitochondrial fluorescence signal changes in HeLa cells expressing indicated mCherry-tagged sspB(LOV2) variants upon photostimulation (470-nm, 30% input). LOVTRAP: Ntom20-Venus-LOV2 and mCherry-Zdk1 (Addgene # 81057). Data are shown as mean ± SD. n = 13 or 15 cells from three independent biological replicates (two-sided unpaired Student’s t-test). S5, LOV-TRAP: P < 0.0001. e Domain architectures of PhoBIT1 constructs used in the study. f Confocal images of a HeLa cell co-expressing mCh-sspB(LOV2) (S5; red) with Ntom20-Venus-ssrA (green) in the absence or presence of photostimulation (470-nm, 30% input). Scale bar, 10 µm. g Quantification of mitochondrial mCherry intensities in PhoBIT1-expressing HeLa cells expressing PhoBIT1 subjected to repeated light-dark cycle stimulation (470-nm, 30% input). The half-lives were determined to be 8.5 s (dissociation) and 28.1 s (re-association), respectively. Data are shown as means ± s.e.m. n = 14 cells from three independent imaging fields. Also see Supplementary Movie 1. h Domain architecture of dCas9-KRAB engineered with PhoBIT1 to enable optogenetic control of the CRISPRi system. i Schematic illustrating light-triggered restoration of the CRISPRi machinery determined by EGFP expression. The ssrA tag is fused to the C-terminus of dCas9, and sspB(LOV2) is fused to the N-terminus of BFP-KRAB. j Confocal images of HeLa cells co-expressing the indicated constructs. Scale bar, 10 µm. k Quantification of EGFP expression in HeLa cells co-transfected with sspB(LOV2)-BFP-KRAB, dCas9-ssrA, and SV40-EGFP, with or without sgNT1; with or without stimulation (470-nm, 30% input). Fluorescence was normalized to the no sgNT1 control in the dark. n = 45 cells from three independent experiments. Data are presented as means ± SD. Statistical significance was determined with two-sided unpaired Student’s t-test. ***P < 0.000001. (Source Data).