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Nucleic Acids Res. 2016 Nov 16;44(20):10003-10014. Epub 2016 Oct 15.

Engineering of temperature- and light-switchable Cas9 variants.

Author information

1
Biophysikalische Chemie, Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany flosopher@gmail.com.
2
Max-Planck-Institute for Infection Biology, 10117 Berlin, Germany.
3
The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå 90187, Sweden.
4
Biophysikalische Chemie, Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany.
5
Biophysikalische Chemie, Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany andreas.moeglich@uni-bayreuth.de.
6
Lehrstuhl für Biochemie, Universität Bayreuth, 95447 Bayreuth, Germany.

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.

PMID:
27744350
PMCID:
PMC5175372
DOI:
10.1093/nar/gkw930
[Indexed for MEDLINE]
Free PMC Article

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