The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.

Active nucleocytoplasmic transport is a key mechanism underlying protein regulation in eukaryotes. While nuclear protein import can be controlled in space and time with a portfolio of optogenetic tools, protein export has not been tackled so far. Here we present a light-inducible nuclear export system (LEXY) based on a single, genetically encoded tag, which enables precise spatiotemporal control over the export of tagged proteins. A constitutively nuclear, chromatin-anchored LEXY variant expands the method towards light inhibition of endogenous protein export by sequestering cellular CRM1 receptors. We showcase the utility of LEXY for cell biology applications by regulating a synthetic repressor as well as human p53 transcriptional activity with light. LEXY is a powerful addition to the optogenetic toolbox, allowing various novel applications in synthetic and cell biology.

Its remarkable ease and efficiency make the CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from adeno-associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components - Cas9 and chimeric g(uide)RNA - either alone or in combination. All our constructs share a modular design that enables simple and stringent guide RNA (gRNA) cloning as well as rapid exchange of promoters driving Cas9 or gRNA. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard-to-transfect targets, as shown for human T-cells. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver-specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR-induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified in the liver of adult mice. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans.