Fig. 1. DiLiCre1.0 system to photo-induce Cre activity.

a Schematic representation of the various photoactivatable Cre recombinase systems: (i) DiLiCre0.0 (adapted from Zhang et al. (2016)), containing the tetracycline response element promoter (TRE3GV) and two flanking PhoCl-ERT2 tandem repeats, and (ii) DiLiCre1.0 construct lacking the N-terminal ERT2-PhoCl tandem repeat. Moreover, schematic representations of the transactivator of the TETon system and the Cre reporter are shown. The Cre reporter is a FLEx vector containing the membrane mTurquoise2 (non-induced) and the nuclear RFP fluorophores (induced). Arrowheads indicate the light-breaking points of the PhoCl fluorophore. b Schematic representation of the mode of action of DiLiCre that recombines the memMTQ2-nRFP reporter. All other DiLiCre systems work in a similar fashion. In brief, doxycycline treated cells express DiLiCre which is sequestered in the cytoplasm by binding to heat-shock protein 90 through the ERT2 domain. As a consequence, the green light emitted from the DiLiCre fluorescent molecule (PhoCl) is observed in the cytoplasm. Upon 405 nm light exposure, PhoCl switches from green-to-red fluorescence, followed by conformational dissociation and cleavage of the fluorophore and subsequently loss of fluorescence. Due to the break, the region containing Cre recombinase dissociates from the ERT2 domain and translocates to the nucleus where it recombines the memMTQ2-nRFP cassette leading to a permanent switch from membranous blue to nuclear red fluorescent. c Representative confocal images of HEK293T (left images) and C26 (right images) cells stably expressing DiLiCre1.0 and memMTQ2-nRFP reporter, before and after photoactivation. The photoactivated region is boxed by blue lines and is mediated by single pulse of laser light at 405 nm (HEK293T: 1.6 mW/mm² for 45-90 s pulse; C26: 7.25 mW/mm² for 45 s). A zoom image of the yellow boxed area is shown at the right of each image. This experiment was independently repeated n = 10 times with similar results. d Time-lapse confocal series of HEK293T (left images) and C26 (right images) cells that have been photoactivated in panel c. A zoom image of the yellow boxed area is shown at the right of each image. Experiments were independently repeated n = 2 times with similar results. The numbers inside the images represent hours and minutes (hh:mm).