Supplemental Information Molecular engineering of single opsins and White-opsin The vector maps of ChR2, ReaChR, and White-opsin are shown in Fig. S1a-c. The gel electrophoresis images of ChR2, ReaChR and White-opsin are shown in Fig. S1d. In Fig. S1eg, we show the absorption spectra of the cloned ChR2, ReaChR and White-opsin plasmids. AmpR(bla) Pbla RSV LTR 5’-R/U5 PBS Psi+ (a) pUC Ori SV40 Early PolyA Blasticidin Resistance (b) hSyn1 RRE EM-7 pLenti-EF1ahChR2-EYFP-WPRE EF1a F1-Ori cPPT hChR2 SV40 PolyA WPRE WPRE (d) Ladder attB3 ChR1 ReaChR attB4 EYFP hChR2 White Opsin E122T E162T VChR1 TS EYFP ReaChR LTR-DeltaU3 Citrine 3’LTR EYFP TETO ReaChR F1-Ori BGH-PolyA LTR-R/U5 pAAV-hSynReaChR-Citrine Amp ChR2 SV40-Ori (c) 5’LTR Citrine CMV pENTR-LaclWhite -Opsin attB1 attR3 Kanamycin T2 T1 pUC Ori (e) (f) (g) 10 15 10 5 0 ReaChR A b s o r p t io n ( a .u .) ChR2 A b s o r p t io n ( a .u .) A b s o r p t io n ( a .u .) 15 10 5 250 275 300 W a v e le n g t h ( n m ) 325 350 6 4 2 0 0 225 White-opsin 8 225 250 275 300 W a v e le n g t h ( n m ) 325 350 225 250 275 300 325 350 W a v e le n g t h ( n m ) Supplementary Figure S1. Molecular engineering of single opsins and White-opsin. (a-c) Vector maps of (a) ChR2, (b) ReaChR, and (c) White-opsin. (d) Gel electrophoresis images of ChR2 (digested by restriction enzymes NotI and BamH1 to fragment sizes of 1.5, 3.3 and 5.6 kb), ReaChR (digested by NotI and BglII to fragment sizes of 3 and 3.3 kb) and White-opsin (digested by EcoRI to fragment sizes of 6.4 and 4.2 kb). (e-g) Absorption spectra of cloned ChR2, ReaChR and White-opsin plasmids. Visualizations of the insertion of White-opsin plasmids into HEK293 cells via optoporation or lipofection are shown in Fig. S2. An increase in intracellular fluorescence (and a few distinct specks attributed to the propidium iodide staining of White-opsin plasmids) after optoporation was observed after optoporation (Fig. S2c). Similarly, propidium-iodide-stained White-opsin plasmids were also observed in the cytoplasm of the lipofected cells (Fig. S2e and S2f). (d) (a) (b) 0 min (e) 25 min (c) 1 min (f) 35 min Supplementary Figure S2. Visualizations of the insertion of White-opsin plasmids into HEK293 cells. (a-c) Optoporation: (a) Bright field and (b) fluorescence images of propidiumiodide-stained White-opsin plasmids in the extracellular medium and (c) the increase in intracellular fluorescence (and a few distinct specks, attributed to the propidium iodide staining of the plasmids) after optoporation (at the site marked by the arrow in a).(d-f) Lipofection: (d) Bright-field image of multiple HEK293 cells and fluorescence images of propidium-iodidestained White-opsin plasmids after (e) 25 min and (f) 35 min of incubation of the cells with the Lipofectamine-plasmid mixture. Scale bar: 5 µm. Fig. S3 shows a representative photocurrent profile induced by white light at an intensity of 0.12 Photocurrent (pA) mW mm-2 (pulse width: 100 ms) in a ChR2-transfected HEK293 cell. 100 ms 0 ChR2 -50 -100 0.0 0.2 0.4 0.6 0.8 1.0 Time (s) Supplementary Figure S3. White-light-induced inward photocurrent in a ChR2transfected HEK293 cell. Representative inward photocurrent profile generated in response to white-light optogenetic stimulation (0.12 mW mm-2) at pulse width of 100 ms. In Fig. S4, we show confocal imaging results for White-opsin-transfected HEK293 cells treated via lipofection. The XY, XZ and YZ cross sections of the White-opsin-expressing cells are shown in Fig. S4a. The cell morphology is observed to be compromised by the transfection process. Fig. S4b-d show the distributions of White-opsin and the nuclear staining dye Hoechst 33242 in live White-opsin-transfected HEK293 cells. XZ (a) XY (b) (c) YZ (d) Supplementary Figure S4. Confocal imaging of HEK293 cells transfected with Whiteopsin via lipofection. (a) The XY, XZ and YZ cross sections of the White-opsin-expressing cells. (b-d) Distributions of (b) White-opsin, (c) the nuclear staining dye Hoechst 33242 in live White-opsin-transfected HEK293 cells and (d) composite image. Scale bar: 5 µm.