title: 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.
authors.affiliation: Biophysikalische Chemie, Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany flosopher@gmail.com.
authors.forename: Florian
authors.initials: F
authors.lastname: Richter
authors.affiliation: Max-Planck-Institute for Infection Biology, 10117 Berlin, Germany.|The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå 90187, Sweden.
authors.forename: Ines
authors.initials: I
authors.lastname: Fonfara
authors.affiliation: Biophysikalische Chemie, Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany.
authors.forename: Boris
authors.initials: B
authors.lastname: Bouazza
authors.affiliation: Biophysikalische Chemie, Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany.
authors.forename: Charlotte Helene
authors.initials: CH
authors.lastname: Schumacher
authors.affiliation: Max-Planck-Institute for Infection Biology, 10117 Berlin, Germany.
authors.forename: Majda
authors.initials: M
authors.lastname: Bratovič
authors.affiliation: Max-Planck-Institute for Infection Biology, 10117 Berlin, Germany.|The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå 90187, Sweden.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Charpentier
authors.affiliation: Biophysikalische Chemie, Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany andreas.moeglich@uni-bayreuth.de.|Lehrstuhl für Biochemie, Universität Bayreuth, 95447 Bayreuth, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Möglich
mesh_terms: D000595:Amino Acid Sequence; D053837:DNA Cleavage / Q000528:radiation effects; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression; D014644:Genetic Variation; D057166:High-Throughput Screening Assays; D008027:Light; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature
chemical_list: D004720:Endonucleases