The assay revealed 43/348 point mutants with improved sensitivity compared to GCaMP3 (ΔF/F0 in response to one action potential; p < 0.01; Wilcoxon rank sum test) (Fig. 1c; Supplementary Fig. 1). In addition, one mutation at the M13/CaM interface (A317E) accelerated kinetics (4-fold), but also reduced response amplitude (2-fold), compared to parent GCaMP variants (Supplementary Fig. 2). In a second round of mutagenesis we combined beneficial mutations, selected based on improved response amplitudes (1-3 action potentials) and/or kinetics, without compromising baseline fluorescence or maximal response (160 action potentials) (94 variants, up to 8 beneficial point mutations; Fig. 1a, c, Supplementary Table 5). In some cases the beneficial effects were additive (Supplementary Fig. 2). A317E consistently accelerated the kinetics compared to the parent sensors. In total, we screened 447 GCaMP variants (Supplementary Table 5).
