(a) Schematics of padCas9-SAM. padCas9-SAM consists of three chimeric proteins, N-terminal fragment of dCas9 (residues 2–713, named dCas9N) fused with pMag (NLS-dCas9N-pMag), C-terminal fragment of dCas9 (residues 714–1368, named dCas9c) fused with VP64 and nMag (nMag-dCas9c-NLS-VP64) and MS2-coat protein fused with p65 and HSF1 activator domains (MS2-NLS-p65-HSF1), and an sgRNA having MS2 aptamers in stem loops, known as sgRNA 2.0. MS2 aptamers in sgRNA recruits MS2-NLS-p65-HSF1, so this system can simultaneously recruit VP64, p65 and HSF1. (b) Schematics of padCas9-Suntag. padCas9-Suntag consists of three chimeric proteins, NLS-dCas9N-pMag, C-terminal fragment of dCas9 fused with nMag and 10 copies of a GCN4 peptide (nMag-dCas9c -NLS-10xGCN4), and single-chain variable fragment nanobody (scFv) for GCN4 fused with VP64 activator domains (scFv-VP64), and an sgRNA. Because scFv-VP64 strongly binds to GCN4 peptides, this system can recruit multiple VP64 into targeted locus by light. (c) Schematics of padCas9-VPR. VPR is a potent tripartite activator consisting of VP64, p65AD and the Epstein-Barr virus R transactivator Rta. padCas9-VPR consists of two chimeric proteins, NLS-dCas9N-pMag and C-terminal fragment of dCas9 fused with nMag and VPR (nMag-dCas9c-NLS-VPR), and an sgRNA. (d) Light-induced endogenous ASCL1 activation in HEK293T cells with padCas9-Suntag, padCas9-VPR and padCas9-SAM with a single sgRNA targeting the promoter region of ASCL1. Data are expressed as relative mRNA amount to the negative control transfected with empty vector in the dark and represented as mean ± s.e.m. (n=3 from three individual experiments). D, Dark; L, Light.