Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein

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.

Enlightening Allostery: Designing Switchable Proteins by Photoreceptor Fusion

“…[90] However, their actual impact on the light switching behavior can be diverse and is hard to predict. [91,92] Interestingly, a recent study from our lab shows that the guidelines above should not be interpreted as being exclusive. Hoffmann et al created light-switchable variants of AcrIIC3, an inhibitor of the high fidelity Cas9 orthologue from Neisseria meningitides (NmeCas9), analogously to CASANOVA.…”

“…[90] Complementary, we and others showed that the exact position within the loop region can significantly affect the light switch. [91,92] Hence, evaluation of several, different insertion variants followed by systematic optimization is usually necessary to obtain a potent light switch.…”

“…Hoffmann et al created light-switchable variants of AcrIIC3, an inhibitor of the high fidelity Cas9 orthologue from Neisseria meningitides (NmeCas9), analogously to CASANOVA. [92] Surprisingly, placing the LOV2 domain into any loops that connected spatially aligned secondary structures did not yield light switching candidates. Instead, inserting the LOV2 into a betasheet, which is part of the NmeCas9 interacting surface of the Acr, resulted in a robust light switch.…”

“…[ 90 ] However, their actual impact on the light switching behavior can be diverse and is hard to predict. [ 91,92 ]…”

CRISPRoff enables spatio-temporal control of CRISPR editing

“…However, once introduced into a cell the CRISPR ribonucleoproteins (RNPs) are uncontrollable, capable of forming double strand breaks (DSBs) at locations where the sgRNA binds within the genome 2 . Previous attempts to control CRISPR systems inside the cell have approached this problem on a variety of levels, from controlled expression of integrated Cas9 3,4 and sgRNA 5 , sgRNAs that are modulated in response to ligand binding 6 , split Cas9 that dimerizes upon illumination with blue light 7 , to anti-CRISPR proteins 8,9 , a viral defense against the CRISPR immune system. However, each of these strategies requires an additional physical component of the CRISPR system beyond a typical RNP to be introduced into the cell.…”