Light-activated Cre recombinase as a tool for the spatial and temporal control of gene function in mammalian cells.
Cre recombinase catalyzes DNA exchange between two conserved lox recognition sites. The enzyme has extensive biological application, from basic cloning to engineering knock-out and knock-in organisms. Widespread use of Cre is due to its simplicity and effectiveness, but the enzyme and the recombination event remain difficult to control with high precision. To obtain such control we report the installation of a light-responsive o-nitrobenzyl caging group directly in the catalytic site of Cre, inhibiting its activity. Prior to irradiation, caged Cre is completely inactive, as demonstrated both in vitro and in mammalian cell culture. Exposure to non-damaging UVA light removes the caging group and restores recombinase activity. Tight spatio-temporal control over DNA recombination is thereby achieved.
Edwards
Wesleigh F
WF
Young
Douglas D
DD
Deiters
Alexander
A
D001665:Binding Sites; D055162:Biocatalysis / Q000528:radiation effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D019426:Integrases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009578:Nitrobenzenes / Q000737:chemistry / Q000494:pharmacology; D011995:Recombination, Genetic / Q000528:radiation effects; D013997:Time Factors; D014466:Ultraviolet Rays
D009578:Nitrobenzenes; D004247:DNA; C045073:Cre recombinase; D019426:Integrases