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Engineering of temp...
Engineering of temperature- and light-switchable Cas9 variants
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Richter, Florian (författare)
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Fonfara, Ines (författare)
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Bouazza, Boris (författare)
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Schumacher, Charlotte Helene (författare)
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Bratovic, Majda (författare)
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Charpentier, Emmanuelle (författare)
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Moeglich, Andreas (författare)
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- 2016
- 2016
- Engelska.
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Ingår i: Nucleic Acids Research. - 0305-1048. ; 44:20, 10003-10014
Abstract
Ämnesord
Stäng
- 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 amechanism 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 Es-cherichia 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 degrees 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.
Ämnesord
- Natural Sciences (hsv)
- Biological Sciences (hsv)
- Biochemistry and Molecular Biology (hsv)
- Naturvetenskap (hsv)
- Biologiska vetenskaper (hsv)
- Biokemi och molekylärbiologi (hsv)
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