ArticleNucleic acids research2016
Engineering of temperature- and light-switchable Cas9 variants.
Article in Nucleic acids research, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 63 papers.
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Who cites it
63 citing papers in PubMed.
- Modular engineering of thermoresponsive allosteric proteins.Nature chemical biology · 2026Article
- Optogenetic Tools for Spatiotemporal Interrogation of Cytoskeletal Dynamics.Bioconjugate chemistry · 2026Review
- Bacteria as living biosensors for DNA.Nature reviews bioengineering · 2026Article
- Red-Light-Only Control of Protein-Protein Interactions Using a Cyanobacteriochrome (UNICYCL).ACS central science · 2026Article
- Physical stimuli-responsive CRISPR-Cas9 systems for spatiotemporally precise control of genome engineering.Theranostics · 2026Review
- Article
- Optogenetic enzymes: A deep dive into design and impact.Current opinion in structural biology · 2025Review
- Engineered bacteria for near-infrared light-inducible expression of cancer therapeutics.Nature cancer · 2025Article
- Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.Nucleic acids research · 2025Article
- Fine-tuning of a CRISPRi screen in the seventh pandemic Vibrio cholerae.BMC genomics · 2024Article
- Intein-based thermoregulated meganucleases for containment of genetic material.Nucleic acids research · 2024Article
- Spatiotemporal control of RNA metabolism and CRISPR-Cas functions using engineered photoswitchable RNA-binding proteins.Nature protocols · 2024Review
- Recent Updates of the CRISPR/Cas9 Genome Editing System: Novel Approaches to Regulate Its Spatiotemporal Control by Genetic and Physicochemical Strategies.International journal of nanomedicine · 2024Review
- Dynamic Metabolic Control: From the Perspective of Regulation Logic.Synthetic biology and engineering · 2023Article
- Light-switchable transcription factors obtained by direct screening in mammalian cells.Nature communications · 2023Article
- Recent Advances in Genome-Editing Technology with CRISPR/Cas9 Variants and Stimuli-Responsive Targeting Approaches within Tumor Cells: A Future Perspective of Cancer Management.International journal of molecular sciences · 2023Review
- CRISPR/Cas9 as a therapeutic tool for triple negative breast cancer: from bench to clinics.Frontiers in molecular biosciences · 2023Review
- CRISPR-Cas-Based Engineering of Probiotics.Biodesign research · 2023Review
- LOV2-based photoactivatable CaMKII and its application to single synapses: Local Optogenetics.Biophysics and physicobiology · 2023Article
- Optogenetics for transcriptional programming and genetic engineering.Trends in genetics : TIG · 2022Review
3 more citing papers are in PubMed but not listed here.
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Authors and funding
7 authors.
Funding
No grant is acknowledged in the PubMed record.
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.