Spatiotemporal control of RNA metabolism and CRISPR-Cas functions using engineered photoswitchable RNA-binding proteins

RNA molecules perform various crucial roles in diverse cellular processes, from translating genetic information to decoding the genome, regulating gene expression and catalyzing chemical reactions. RNA-binding proteins (RBPs) play an essential role in regulating the diverse behaviors and functions of RNA in live cells, but techniques for the spatiotemporal control of RBP activities and RNA functions are rarely reported yet highly desirable. We recently reported the development of LicV, a synthetic photoswitchable RBP that can bind to a specific RNA sequence in response to blue light irradiation. LicV has been used successfully for the optogenetic control of RNA localization, splicing, translation and stability, as well as for the photoswitchable regulation of transcription and genomic locus labeling. Compared to classical genetic or pharmacologic perturbations, LicV-based light-switchable effectors have the advantages of large dynamic range between dark and light conditions and submicron and millisecond spatiotemporal resolutions. In this protocol, we provide an easy, efficient and generalizable strategy for engineering photoswitchable RBPs for the spatiotemporal control of RNA metabolism. We also provide a detailed protocol for the conversion of a CRISPR-Cas system to optogenetic control. The protocols typically take 2-3 d, including transfection and results analysis. Most of this protocol is applicable to the development of novel LicV-based photoswitchable effectors for the optogenetic control of other RNA metabolisms and CRISPR-Cas functions. This protocol provides an efficient and generalizable strategy for engineering photoswitchable RNA-binding proteins (RBPs) for the spatiotemporal control of RNA activity. It uses LicV, a synthetic RBP that can bind to a specific RNA sequence in response to blue light irradiation.This optogenetic method circumvents the limitations of previous strategies by enabling the activity of functional RNAs or effectors to be more precisely controlled because they can be switched on and off by using light. This protocol for the spatiotemporal control of RNA activity uses LicV, a synthetic, photoswitchable RNA-binding protein (RBP) that can bind to a specific RNA sequence in response to blue light irradiation, and provides an efficient and generalizable strategy for engineering photoswitchable RBPs.