Figure 6.

Use of sunbody to enable photoactivatable gene transcription and base editing. Two-way ANOVA Tukey’s multiple comparison test was used for statistical analysis, **** p < 0.0001. Data are shown as mean ± SEM. a) Cartoon illustrating the combination of sunbody with a modified FLARE system (designated SolarFLARE) to enable light-inducible expression of genes of interest, such as TagBFP as a reporter or the N-terminal domain of MLKL (MLKL-NT) as a necroptosis inducer. b) Quantification of BFP expression in HeLa cells transfected with SolarFLARE (sunbody-TEV + FLARE) or the control (sunbody alone + FLARE) vectors, as well as the TagBFP reporter gene, before and after light illumination for 8 h. n = 10 fields of view from three independent assays. c) Quantification of necroptotic cell death as indicated by SYTOX blue nuclear staining of dead cells. HeLa cells were transfected with SolarFLARE (sunbody-TEV + FLARE) or the control (sunbody alone + FLARE) vectors, as well as the inducible MLKL-NT expression cassette, before and after light illumination for 8 h. Also see Figure S4, Supporting Information, for representative images. n = 10 fields of view from three independent assays. d) Design of a photoactivatable cytosine base editor (paCBE). Upon photostimulation, sunbody-mCh association reassembles two functional units of CBE (Part I: the mCh-Cas9n/sgRNA for genome targeting; Part II: APOBEC1-sunbody-UGI for C-to-T conversion) to restore the activity of paCBE. A “Gene ON” (GO) luciferase reporter system is used to report the activity of paCBE before and after light stimulation. Successful recruitment of Part II to the targeted genomic locus is anticipated to cause C-to-T conversion in the start codon (ACG > ATG) to initiate the translation of a luciferase reporter gene. e) Quantification of the base editing efficiency of paCBE by using luciferase activity as readout. Sunbody alone was used as a negative control. n = 3 independent assays.