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.

Analysis of Physicochemical Properties, Lipid Composition, and Oxidative Stability of Cashew Nut Kernel Oil.

Cashew nut kernel oil (CNKO) is an important oil source from tropical crops. The lipid species, composition, and relative content of CNKO were revealed using ultra high performance liquid chromatography time-of-flight tandem mass spectrometry (UPLC-TOF-MS/MS), and the physicochemical properties, functional group structure, and oxidation stability of CNKO at different pressing temperatures were characterized using a near infrared analyzer and other methods. The results showed that CNKO mainly consisted of oleic acid (60.87 ± 0.06%), linoleic acid (17.33 ± 0.28%), stearic acid (10.93 ± 0.31%), and palmitic acid (9.85 ± 0.04%), and a highly unsaturated fatty acid (78.46 ± 0.35%). In addition, 141 lipids, including 102 glycerides and 39 phospholipids, were identified in CNKO. The pressing temperature had a significant effect on the physicochemical properties of cashew kernels, such as acid value, iodine value, and peroxide value, but the change in value was small. The increase in pressing temperature did not lead to changes in the functional group structure of CNKO, but decreased the induction time of CNKO, resulting in a decrease in their oxidative stability. It provided basic data support to guide subsequent cashew kernel processing, quality evaluation, and functional studies.