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- Richter, Florian, et al.
(författare)
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Engineering of temperature- and light-switchable Cas9 variants
- 2016
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 44:20, s. 10003-10014
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Tidskriftsartikel (refereegranskat)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 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 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 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.
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| 2. |
- Richter, Florian, et al.
(författare)
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Switchable Cas9
- 2017
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Ingår i: Current Opinion in Biotechnology. - : CURRENT BIOLOGY LTD. - 0958-1669 .- 1879-0429. ; 48, s. 119-126
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Forskningsöversikt (refereegranskat)abstract
- Ever since its discovery, Cas9 from Streptococcus pyogenes has revolutionized biology by enabling analysis and engineering of genomes with unprecedented precision and ease. To fine-tune on-target effects and to mitigate adverse effects caused by untimely and off-target action of Cas9, strategies have been developed to control its activity at the post-translational stage via external trigger signals. Control is either achieved by modifying the Cas9 protein itself or its programmable RNA molecules. To date, switchable Cas9 variants responding to small ligands, light or temperature have been engineered. With these variants in hand, the regulation and modification of genomes can be accomplished in graded and ever more precise manner.
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