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.
Highlights
Genome engineering technologies based on CRISPR–Cas systems facilitate site-specific targeting and manipulation of genes in living cells [1,2,3] and currently transform many areas of biomedical research
We report the engineering and application of CASANOVA-C3, a lightdependent anti-CRISPR protein for conditional activation of NmeCas9
Excitation with blue light triggers the unfolding and undocking of the J␣ and Avena sativa (As)’␣ helices, resulting in a massive gain of flexibility at the LOV2 termini [63,64]. It has previously been shown by Klaus Hahn et al that inserting the AsLOV2 domain into surface-exposed loops of enzymes can be used to disrupt their function in a light-dependent manner [65]
Summary
Genome engineering technologies based on CRISPR (clustered regularly-interspaced short palindromic repeats)–Cas systems facilitate site-specific targeting and manipulation of genes in living cells [1,2,3] and currently transform many areas of biomedical research. The class 2 type-II effectors, typically applied for genome engineering, comprise a Cas nuclease as the protein component and a single guide RNA (sg)RNA, which directs the Cas nuclease to selected nucleic acid targets by means of sequence complementarity Due to their simplicity and versatility, class 2 CRISPR systems enable a plethora of applications including targeted induction of DNA doublestrand breaks for genome editing [1,2,5], regulation of endogenous transcription [5,6], epigenetic reprogramming [7,8,9], DNA labeling [10,11] and base editing [12,13]. Due to its large size of 1,368 amino acids
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