KillerOrange represents a mutant of KillerRed with a bright orange fluorescence (excitation maximum at 512 nm and emission maximum at 555 nm) [Sarkisyan et al., 2015]. In contrast to KillerRed, which becomes phototoxic after illumination with green or orange (540-580 nm) light, KillerOrange was shown to be phototoxic to E.coli after illumination with blue or green (450-540 nm) light.

The blue-shifted spectrum of KillerOrange makes it potentially more suitable for two-photon microscopy than the parental KillerRed. Also, the large Stokes shift of over 40 nm should make it possible to spectrally separate signals of KillerOrange from cyan and green fluorescent proteins when the proteins are excited simultaneously by blue light. One can thus use green and cyan indicators to observe the effects of phototoxicity in real time without the need to change the excitation light.

KillerOrange-KillerRed pair can potentially be used to independently ablate two cell populations. This pair also promises the orthogonal optical control of the propagation of signaling cascades either by chromophore-assisted light inactivation of the participating proteins or by triggering cascades with hydrogen peroxide produced by KillerRed and likely by KillerOrange. KillerOrange-KillerRed tandem fusions or combination of various photosensitizers in one cassette may enhance phototoxicity under white light irradiation and may be useful as a research tool in biology.