(a) Optimizing subcellular localization of padCas9 fragments and replacing nMag photoactivatable dimerization domain. The nuclear localization signal (NLS) of NLS-dCas9N-pMag was removed and two copies of nuclear export signal (NES) were fused with dCas9N-pMag (NES-dCas9N-pMag-NES). We also replaced nMag of nMag-dCas9C-NLS-VP64 with nMagHigh1 for enhancing dimerization efficiency (nMagHigh1-dCas9C-NLS-VP64). (b) Screening of MS2 effectors that have different designs. About MS2-coat protein, we tested MS2(N55K) mutant, which is originally used in SAM, and non-aggregating MS2 delta FG mutant. We also tested whether the number and position of NLS effects induction potency. (c) Gene activation efficiencies of several MS2 effectors with NES-dCas9N-pMag-NES and nMagHigh1-dCas9C-NLS-VP64. sgRNA 2.0 targeting human ASCL1 is used. Data are expressed as relative ASCL1 mRNA amount to the negative control transfected with empty vector in the dark and represented as mean ± s.e.m. (n=4 from two individual experiments with two cell culture replicates). D, Dark; L, Light.