Engineering of temperature- and light-switchable Cas9 variants

Titelangaben

Richter, Florian ; Fonfara, Ines ; Bouazza, Boris ; Schumacher, Charlotte Helene ; Bratovič, Majda ; Charpentier, Emmanuelle ; Möglich, Andreas:
Engineering of temperature- and light-switchable Cas9 variants.
In: Nucleic Acids Research. Bd. 44 (2016) Heft 20 . - S. 10003-10014.
ISSN 1362-4962
DOI: https://doi.org/10.1093/nar/gkw930

Volltext

Link zum Volltext (externe URL):

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.