CreLite: An Optogenetically Controlled Cre/loxP System Using Red Light

Abstract

Precise manipulation of gene expression with temporal and spatial control is essential for functional studies and the determination of cell lineage relationships in complex biological systems.The Cre-loxP system is commonly used for gene manipulation at desired times and places.However, specificity is dependent on the availability of tissue-or cell-specific regulatory elements used in combination with Cre or CreER (tamoxifen-inducible). Here we present CreLite, an optogenetically-controlled Cre system using red light in developing zebrafish embryos. Cre activity is disabled by splitting Cre and fusing the inactive halves with the Arabidopsis thaliana red light-inducible binding partners, PhyB and PIF6. In addition, PhyB-PIF6 binding requires phycocyanobilin (PCB), providing an additional layer of control. Upon exposure to red light (660 nm) illumination, the PhyB-CreC and PIF6-CreN fusion proteins come together in the presence of PCB to restore Cre activity. Red-light exposure of transgenic zebrafish embryos harboring a Credependent multi-color fluorescent protein reporter (ubi:zebrabow) injected with CreLite mRNAsand PCB, resulted in Cre activity as measured by the generation of multi-spectral cell labeling in various tissues, including heart, skeletal muscle and epithelium. We show that CreLite can be used for gene manipulations in whole embryos or small groups of cells at different stages of development. CreLite provides a novel optogenetic tool for precise temporal and spatial control of gene expression in zebrafish embryos that may also be useful in cell culture, ex vivo organ culture, and other animal models for developmental biology studies.

A far-red light–inducible CRISPR-Cas12a platform for remote-controlled genome editing and gene activation

“…Red/FRL-responsive optogenetic devices reported to date have been based on PhyB/PIF6 derived from Arabidopsis thaliana ( 53 , 54 ) and BphP1/PpsR2 (or an engineered QPAS1) derived from Rhodopseudomonas palustris ( 55 , 56 ); there is also a recently reported near-infrared optogenetic system based on IsPadC-PCM from Idiomarina sp. A28L ( 57 ).…”

Rebuilding limbs, one cell at a time

“…The development of new optogenetic transgenic tools is also a promising means to manipulate cells during the process of regeneration. Light‐controlled transgenes could be used as a means to simply label cells with high temporal and spatial precision using photoactivatable fluorophores or light‐inducible Cre recombinase 55 . Other sensors would have the ability to perturb intracellular pathways required for cell behaviors and fate decisions 56 .…”

The Red Edge: Bilin-Binding Photoreceptors as Optogenetic Tools and Fluorescence Reporters

“…Another sophisticated PhyB/PIF6 system was designed by Yen et al for light-controlled DNA manipulation . The authors brought the Cre/ lox P function under red light control.…”