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.

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upon photoexcitation and often reversible at similar time-scales upon withdrawal of the light trigger. [1][2][3][4] Apart from this temporal precision, light can also be supplied in a spatially confined way with single-cell or subcellular accuracy, for example by using lasers or blinds. [5][6][7] Together, these favorable characteristics render optogenetics a highly powerful method for perturbing and studying dynamic processes in living cells and explain the rapid growth of this scientific field over the past 15 years.Generally, optogenetic tools can either be categorized by the nature of the photoreceptors they employ or by their undelaying working principle. [8] Focusing on the latter classification, one can differentiate between systems that are regulated via inducible association/dissociation reactions and often involve multiple component versus single-component tools functioning via inducible allostery. [8] Association-based tools make use of photoreceptors that bind to one another or to a secondary binding partner upon light exposure. [9] The red/far-red light-responsive PhyB/PIF or blue light-responsive CRY2/CIB1 heterodimerization systems are typical examples for association-dependent optogenetic tools. One application of such optogenetic heterodimerizers is the control of protein subcellular localization. [10] To this end, one binding partner is targeted to a specific subcellular structure or location (e.g., the plasma membrane, nucleus, mitochondria), while the second partner is corecruited to this structure upon dimerization. The same principle can also be employed to reconstitute split-proteins in a light-dependent manner. [9] Optogenetic tools that function via inducible allostery, in contrast, are usually single-component systems. They consist of an engineered chimera between a photoreceptor domain and an effector protein and their light-switching behavior is mediated by steric or allosteric interactions between the two. [8,11] A particular advantage of single-component optogenetic tools as compared to dimerization-based tools is their compactness. They are small in size, since only the photosensory domain is fused to the effector domain/protein of choice and no additional proteins need to be coexpressed. Moreover, allosteric tools do not rely on the diffusion-based association of two components, the kinetics of which depends on the protein concentrations.Optogenetic switches that rely on inducible allostery have mainly been engineered on the basis of light-oxygen-voltage (LOV) domains [11] and phytochromes. [12][13][14] LOV domains belong to the Per-ARNT-Sim or PAS (period circadian protein-aryl 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-componen...