Engineering strategy and vector library for the rapid generation of modular light-controlled protein-protein interactions

Abstract

Optogenetics enables the spatio-temporally precise control of cell and animal behaviour. Many optogenetic tools are driven by light-controlled protein-proteininteractions (PPIs) that are repurposed from natural light-sensitive domains (LSDs).Applying light-controlled PPI to new target proteins is challenging because it is difficult to predict whether one the many available LSDs will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of lightcontrolled PPIs. The strategy permits fusing a target protein to LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes.Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.

Optogenetic control of apical constriction induces synthetic morphogenesis in mammalian tissues

“…For OptoShroom3 design, mouse Shroom3 gene was used, which was a gift from T. Nishimura, from M. Takeichi lab. Construction of OptoShroom3 was carried out using iLID and Sspb genes from the optogenetic vector library published by Tichy and colleagues 51 , which was a gift from H. Janovjack lab. OptoShroom3 sequences can be found in supplementary table 1. iRFP-CAAX and GFP-CAAX sequences were prepared through traditional cloning methods.…”