Engineering of temperature- and light-switchable Cas9 variants

Abstract

Sensory photoreceptors have enabled non-invasive and spatiotemporal control of numerous biological processes. Photoreceptor engineering has expanded the repertoire beyond natural receptors, but to date no generally applicable strategy exists towards constructing light-regulated protein actuators of arbitrary function. We hence explored whether the homodimeric Rhodobacter sphaeroides light-oxygen-voltage (LOV) domain (RsLOV) that dissociates upon blue-light exposure can confer light sensitivity onto effector proteins, via a mechanism of light-induced functional site release. We chose the RNA-guided programmable DNA endonuclease Cas9 as proof-of-principle effector, and constructed a comprehensive library of RsLOV inserted throughout the Cas9 protein. Screening with a high-throughput assay based on transcriptional repression in Escherichia coli yielded paRC9, a moderately light-activatable variant. As domain insertion can lead to protein destabilization, we also screened the library for temperature-sensitive variants and isolated tsRC9, a variant with robust activity at 29°C but negligible activity at 37°C. Biochemical assays confirmed temperature-dependent DNA cleavage and binding for tsRC9, but indicated that the light sensitivity of paRC9 is specific to the cellular setting. Using tsRC9, the first temperature-sensitive Cas9 variant, we demonstrate temperature-dependent transcriptional control over ectopic and endogenous genetic loci. Taken together, RsLOV can confer light sensitivity onto an unrelated effector; unexpectedly, the same LOV domain can also impart strong temperature sensitivity.

Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein

“…Systems to confine the activity of Cas9 in time and space are highly desired, as they improve the precision at which CRISPR-mediated genome perturbations can be made (25,26,30). We had previously engineered CASANOVA, an optogenetic SpyCas9 inhibitor based on LOV2 insertion into AcrIIA4 (29).…”

“…The ability to control and fine-tune NmeCas9 activity via exogenous stimuli would further enhance the precision at which CRISPR genome perturbations can be made. Unlike SpyCas9, for which a whole battery of tools exist that facilitate its conditional activation by chemical triggers (21)(22)(23)(24), light (25)(26)(27)(28)(29) or temperature (30,31), no method for conditional activation of NmeCas9 by exogenous triggers has yet been developed.…”

High-performance chemical and light-inducible recombinases in mammalian cells and mice

“…Temperature induction was performed by exposing the cells to either room temperature (22°C) or refrigerated (4°C) conditions for four hourly 15-minute pulses 18 hours after transfection. The temperature sensitivity of this system may not be completely surprising as VVD has been found to play a role in temperature regulation 28,29 and others have engineered another closely related blue LID system to be more sensitive to colder temperature 30 .…”

Engineering genetically-encoded tools for optogenetic control of protein activity

“…Similarly, light-dependent sequestering and inactivation was also achieved by fusing target proteins to CRY2olig, a mutant of CRY2 that forms large clusters on its own and thus does not require co-expression of the CIB1-multimeric protein [18]. In contrast to using photoreceptors that oligomerize with light to disrupt activity, a recent study explored the promise of light-induced monomerization to stimulate activity [50]. In this work, the LOV domain from Rhodobacter sphaeroides ( Rs LOV), which dissociates into monomers upon blue light stimulation, was inserted in solvent-exposed regions of Cas9 to disrupt activity through dark dimerization.…”