Photoswitchable gRNAs for spatiotemporally controlled CRISPR-Cas-based genomic regulation
File(s)2020-Omori-ACSCS-accepted.pdf (1.81 MB)
Accepted version
Author(s)
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.
Date Issued
2020-05-27
Date Acceptance
2020-03-25
Citation
ACS Central Science, 2020, 6 (5), pp.695-703
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
Medical Research Council (MRC)
British Heart Foundation
Grant Number
MR/R015651/1
RE/18/4/34215
Subjects
03 Chemical Sciences
Publication Status
Published
Date Publish Online
2020-04-01