. Electroporation-Based Screening of GEVIs (A) Left: responses of various GEVIs in HEK293-Kir2.1 cells to a 0.01-ms 150-V pulse recorded at 100 Hz. ASAP1ncp is a voltage-insensitive variant of ASAP1 in which the GFP was de-permuted (Chamberland et al., 2017). Solid traces, mean responses. Data are from five or six wells from two separate experiments, with thetopfiverespondersineachwellanalyzed.Right:fluorescenceresponseswererecordedat200HzinHEK293AcellsexpressingvariousGEVIsinresponsetoa voltage step from 70 to 0 mV (ASAP1ncp, n = 4; ASAP1, n = 6; Arclight, n = 3; ASAP2s, n = 10). Error bars, standard deviations (SDs). (B) ASAP can be directly expressed from these unpurified PCR products. HEK293-Kir2.1 cells were transiently transfected using Lipofectamine 3000 ａnd either pc3-CMV-ASAP2s plasmid or linear ASAP2s product generated by PCR. Intensity scaling is the same across images. (C) Overview of the screening system. HEK293-Kir2.1 cells expressing GEVI variants are plated in 384-well plates on conductive glass slides connected to a square-pulse generator. The medium in each well is sequentially contacted by a motorized platinum electrode during imaging by a high-speed CMOS camera. The entire procedure is automated by MATLAB routines. (D) Left: model of ASAP domain organization. Right: residues targeted for mutagenesis in the S3-cpGFP linker. (E) Evolutionary history of ASAP3. (F) Fluorescence responses from the third round of electrical screening for the parental ASAP2f L146G S147T R414Q (left) ａnd the best-performing mutant, ASAP2f L146G S147T S150G H151D R414Q (right). (G) One-photon fluorescence response curves of ArcLight Q239 (n = 3), ASAP2f R414Q (n = 6), ａnd ASAP3 (n = 10) from HEK293 cells stepped for 500 ms from a holding potential of 70 mV. Error bars, SEMs. See also Figures S1, S2, S3, ａnd S4.