Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light-Activated Guide RNA |
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Authors: | Wenyuan Zhou Wes Brown Anirban Bardhan Dr Michael Delaney Amber S Ilk Randy R Rauen Dr Shoeb I Kahn Prof?Dr Michael Tsang Prof?Dr Alexander Deiters |
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Institution: | 1. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, 15260 USA;2. Horizon Discovery, 2650 Crescent Drive, Lafayette, CO, 80026 USA;3. Department of Developmental Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, 15260 USA |
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Abstract: | We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5′-protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA-target hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off-to-on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation. |
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Keywords: | CRISPR/Cas9 gene technology optical control photocaged compounds RNA |
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