posted on 2020-03-31, 20:29authored byElena
V. Moroz-Omori, Dwiantari Satyapertiwi, Marie-Christine Ramel, Håkon Høgset, Ilona K. Sunyovszki, Ziqian Liu, Jonathan P. Wojciechowski, Yueyun Zhang, Christopher L. Grigsby, Liliana Brito, Laurence Bugeon, Margaret J. Dallman, Molly M. Stevens
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.