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Photoswitchable gRNAs for spatiotemporally controlled CRISPR-Cas-based genomic regulation

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Title: Photoswitchable gRNAs for spatiotemporally controlled CRISPR-Cas-based genomic regulation
Authors: Moroz-Omori, EV
Satyapertiwi, D
Ramel, MC
Hogset, H
Sunyovszki, I
Liu, Z
Wojciechowski, J
Zhang, Y
Grigsby, CL
Casimiro Brito, L
Bugeon, L
Dallman, M
Stevens, M
Item Type: Journal Article
Abstract: The recently discovered CRISPR-Cas gene editing system and its derivatives have found numerous applications in fundamental biology research and pharmaceutical sciences. The need for precise external control over the gene editing and regulatory events has driven the development of inducible CRISPR-Cas systems. While most of the light-controllable CRISPR-Cas systems are based on protein engineering, we developed an alternative synthetic approach based on modification of crRNA/tracrRNA duplex (guide RNA or gRNA) with photocaging groups, preventing the gRNA from recognizing its genome target sequence until its deprotection is induced within seconds of illumination. This approach relies on a straightforward solid-phase synthesis of the photocaged gRNAs, with simpler purification and characterization processes in comparison to engineering a light-responsive protein. We have demonstrated the feasibility of photocaging of gRNAs and light-mediated DNA cleavage upon brief exposure to light in vitro. We have achieved light-mediated spatiotemporally resolved gene editing as well as gene activation in cells, whereas photocaged gRNAs showed virtually no detectable gene editing or activation in the absence of light irradiation. Finally, we have applied this system to spatiotemporally control gene editing in zebrafish embryos in vivo, enabling the use of this strategy for developmental biology and tissue engineering applications.
Issue Date: 27-May-2020
Date of Acceptance: 25-Mar-2020
URI: http://hdl.handle.net/10044/1/79025
DOI: 10.1021/acscentsci.9b01093
ISSN: 2374-7943
Publisher: American Chemical Society
Start Page: 695
End Page: 703
Journal / Book Title: ACS Central Science
Volume: 6
Issue: 5
Copyright Statement: © 2020 American Chemical Society. . This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Central Science, after peer review and technical editing by the publisher. To access the final edited and published work see https://pubs.acs.org/doi/10.1021/acscentsci.9b01093
Sponsor/Funder: Medical Research Council (MRC)
British Heart Foundation
Funder's Grant Number: MR/R015651/1
Keywords: 03 Chemical Sciences
Publication Status: Published
Online Publication Date: 2020-04-01
Appears in Collections:Materials
National Heart and Lung Institute
Faculty of Medicine
Faculty of Natural Sciences
Faculty of Engineering