CreLite: An optogenetically controlled Cre/loxP system using red light

Abstract

Background: Precise manipulation of gene expression with temporal and spatial control is essential for functional analysis and determining cell lineage relationships in complex biological systems. The cyclic recombinase (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. Here, we present CreLite, an optogenetically controlled Cre system using red light in developing zebrafish embryos. Results: Cre activity is disabled by splitting Cre and fusing with the Arabidopsis thaliana red light-inducible binding partners, PhyB and PIF6. Upon red light illumination, the PhyB-CreC and PIF6-CreN fusion proteins come together in the presence of the cofactor phycocyanobilin (PCB) to restore Cre activity. Red light exposure of zebrafish embryos harboring a Cre-dependent multicolor fluorescent protein reporter injected with CreLite mRNAs and PCB resulted in Cre activity as measured by the generation of multispectral cell labeling in several different tissues. Conclusions: Our data show that CreLite can be used for gene manipulations in whole embryos or small groups of cells at different developmental stages, and suggests CreLite may also be useful for temporal and spatial control of gene expression in cell culture, ex vivo organ culture, and other animal models.

Stable Transgenic Mouse Strain with Enhanced Photoactivatable Cre Recombinase for Spatiotemporal Genome Manipulation

“…Among them, the red‐light‐dependent Cre‐loxP system (CreLite) enables multispectral cell labeling in zebrafish embryos. [ 9 ] However, CreLite, based on the PhyB‐PIF6 system, requires the addition of an exogenous phycocyanobilin chromophore, which limits its in vivo applicability, especially in higher animals. Blue light‐induced dimerization modules, such as cryptochrome 2 (CRY2), magnets, vivid (VVD), and AsLOV2, have been used to develop photoactivatable Cre recombinases without the need for exogenous chromophores.…”

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 ).…”

Optically Controlled CRISPR-Cas9 and Cre Recombinase for Spatiotemporal Gene Editing: A Review

“…Red-light-activated split-Cre systems overcome the disadvantages of using shorter-wavelength blue and UV light (e.g., phototoxicity and low tissue penetration) and can be used for applications requiring greater tissue penetration in vivo . ,, L-SCRaMbLE and CreLite are two such systems based on the Phytochrome B (PhyB) and Phytochrome interacting factor 6 (PIF6) photodimerization system and require phycocyanobilin (PCB) as an exogenous chromophore. L-ScRaMbLE comprises the CreN fragment fused to PhyB, and the CreC fragment fused to PIF6 and a nuclear localization sequence (NLS), to create a physical separation between the two halves.…”