Curated Optogenetic Publication Database

Abstract: Optogenetic control of CRISPR-Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and engineered, the need for straightforward strategies to control their activity via exogenous stimuli persists. The Cas9 from Neisseria meningitidis (Nme) is a particularly small and target-specific Cas9 orthologue, and thus of high interest for in vivo genome editing applications. Here, we report the first optogenetic tool to control NmeCas9 activity in mammalian cells via an engineered, light-dependent anti-CRISPR (Acr) protein. Building on our previous Acr engineering work, we created hybrids between the NmeCas9 inhibitor AcrIIC3 and the LOV2 blue light sensory domain from Avena sativa. Two AcrIIC3-LOV2 hybrids from our collection potently blocked NmeCas9 activity in the dark, while permitting robust genome editing at various endogenous loci upon blue light irradiation. Structural analysis revealed that, within these hybrids, the LOV2 domain is located in striking proximity to the Cas9 binding surface. Together, our work demonstrates optogenetic regulation of a type II-C CRISPR effector and might suggest a new route for the design of optogenetic Acrs.

Abstract: Optogenetics harnesses natural photoreceptors to non-invasively control selected processes in cells with previously unmet spatiotemporal precision. Linking the activity of a protein of choice to the conformational state of a photosensor domain through allosteric coupling represents a powerful method for engineering light-responsive proteins. It enables the design of compact and highly potent single-component optogenetic systems with fast on- and off-switching kinetics. However, designing protein-photoreceptor chimeras, in which structural changes of the photoreceptor are effectively propagated to the fused effector protein, is a challenging engineering problem and often relies on trial and error. Here, recent advances in the design and application of optogenetic allosteric switches are reviewed. First, an overview of existing optogenetic tools based on inducible allostery is provided and their utility for cell biology applications is highlighted. Focusing on light-oxygen-voltage domains, a widely applied class of small blue light sensors, the available strategies for engineering light-dependent allostery are presented and their individual advantages and limitations are highlighted. Finally, high-throughput screening technologies based on comprehensive insertion libraries, which could accelerate the creation of stimulus-responsive receptor-protein chimeras for use in optogenetics and beyond, are discussed.

Abstract: Since the breakthrough discoveries that CRISPR-Cas9 nucleases can be easily programmed and employed to induce targeted double-strand breaks in mammalian cells, the gene editing field has grown exponentially. Today, CRISPR technologies based on engineered class II CRISPR effectors facilitate targeted modification of genes and RNA transcripts. Moreover, catalytically impaired CRISPR-Cas variants can be employed as programmable DNA binding domains and used to recruit effector proteins, such as transcriptional regulators, epigenetic modifiers or base-modifying enzymes, to selected genomic loci. The juxtaposition of CRISPR and optogenetics enables spatiotemporally confined and highly dynamic genome perturbations in living cells and animals and holds unprecedented potential for biology and biomedicine.Here, we provide an overview of the state-of-the-art methods for light-control of CRISPR effectors. We will detail the plethora of exciting applications enabled by these systems, including spatially confined genome editing, timed activation of endogenous genes, as well as remote control of chromatin-chromatin interactions. Finally, we will discuss limitations of current optogenetic CRISPR tools and point out routes for future innovation in this emerging field.