Structures of Neisseria meningitidis Cas9 Complexes in Catalytically Poised and Anti-CRISPR-Inhibited States

Sun, Wei; Yang, Jing; Cheng, Zhi; Amrani, Nadia; Liu, Chao; Wang, Kangkang; Ibraheim, Raed; Edraki, Alireza; Huang, Xue; Wang, Min; Wang, Jiuyu; Liu, Liang; Sheng, Gang; Yang, Yanhua; Lou, Jizhong; Sontheimer, Erik J.; Wang, Yanli

doi:10.1016/j.molcel.2019.09.025

Cited by 95 publications

(133 citation statements)

References 56 publications

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“…Figure S7). In some cases, these differences were even greater as was noted for orthologs from Neisseria meningitidis (Nme) where PI domains only shared 52% identical residues while the rest of the protein was nearly the same (98% identity) 56 Figure S12). Altogether, these observations could in part be explained by the uncoupling of PI domain evolution from the rest of protein indicating that it is under selective pressure to diversify perhaps in response to PAM-based phage escape strategies as described previously [57][58][59] .…”

Section: Discussionmentioning

confidence: 87%

Biochemically diverse CRISPR-Cas9 orthologs

Gasiūnas

Young

Karvelis

et al. 2020

Preprint

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CRISPR-Cas9 nucleases are abundant in microbes. To explore this largely uncharacterized diversity, we applied cell-free biochemical screens to rapidly assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of novel Cas9 proteins. This approach permitted the characterization of 79Cas9 orthologs with at least 7 distinct classes of gRNAs and 50 different PAM sequence requirements.PAM recognition spanned the entire spectrum of T-, A-, C-, and G-rich nucleotides ranging from simple di-nucleotide recognition to complex sequence strings longer than 4. Computational analyses indicated that most of this diversity came from 4 groups of interrelated sequences providing new insight into Cas9 evolution and efforts to engineer PAM recognition. A subset of Cas9 orthologs were purified and their activities examined further exposing additional biochemical diversity. This constituted both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for longer stretches of homology between gRNA and DNA target to function robustly. In all, the diverse collection of Cas9 orthologs presented here sheds light on Cas9 evolution and provides a rich source of PAM recognition and other potentially desirable properties that may be mined to expand the genome editing toolbox with new RNA-programmable nucleases.

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Section: Discussionmentioning

confidence: 87%

Biochemically diverse CRISPR-Cas9 orthologs

Gasiūnas

Young

Karvelis

et al. 2020

Preprint

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“…When developing CASANOVA, we relied on the available structural information, which guided our selection of LOV2 insertion sites on AcrIIA4. The AcrIIC3 structure, however, was not known at the start of this project and has only been reported very recently (48,61,62). Thus, we started this work by performing a secondary structure prediction using QUARK (63) with the goal to roughly identify regions corresponding to alpha-helices, beta-sheets and unstructured loops, as the latter are more permissive to domain insertions.…”

Section: Resultsmentioning

confidence: 99%

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

Hoffmann

Mathony

Belzen

et al. 2019

Preprint

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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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“…To date, inhibition of target binding is the prevalent strategy. Thirteen anti-CRISPR proteins interfere with target recognition and binding (type I-F AcrIF1, AcrIF2 and AcrIF10 [34][35][36][37]; type II-A AcrIIA2, AcrIIA4, AcrIIA5 and AcrIIA6 [38][39][40][41][42][43][44][45][46][47]; type II-C AcrIIC3, AcrIIC4 and AcrIIC5 [48][49][50][51]; type V-A AcrVA1, AcrV4A and AcrVA5 [52][53][54][55][56][57][58]), while only five-block target cleavage (type I-E AcrIE1 [29,59]; type III-B AcrIIIB1 [12]; type I-F AcrIF3 [26,27]; type II-C AcrIIC1 and AcrIIC3 [48][49][50]) ( Figure 1). Given that DNA binding is the ratelimiting step of Cascade and Cas9-mediated interference activities [60,61], altering this step is, therefore, an efficient way to inactivate CRISPR-Cas interference.…”

Section: Inhibition Of Crispr-cas Interferencementioning

confidence: 99%

“…As for AcrIIC3, one monomer is able to tether two NmeCas9 effector complexes ( Figure 2B). It interacts with the NmeCas9 HNH domain of one effector complex, at the opposite face of the catalytic site, and the NmeCas9 REC lobe (glossary) of another effector complex [33,50,66]. The interaction between AcrIIC3 and the REC lobe, in the vicinity of the DNA-sgRNA hybridization site, likely perturbs the conformational dynamics of NmeCas9 that assists DNA binding, which would explain the reduction in NmeCas9 DNA binding affinity in vitro and the inhibition of DNA binding within cells [48,49].…”

Section: Allosteric Inhibition and Clustering Of Effector Complexesmentioning

confidence: 99%

“…The interaction between AcrIIC3 and the REC lobe, in the vicinity of the DNA-sgRNA hybridization site, likely perturbs the conformational dynamics of NmeCas9 that assists DNA binding, which would explain the reduction in NmeCas9 DNA binding affinity in vitro and the inhibition of DNA binding within cells [48,49]. Besides, AcrIIC3 is able to associate with DNA-bound NmeCas9 effector complexes in vitro [50]. The interaction between AcrIIC3 and the HNH domain blocks the structural changes triggered by target DNA binding, and therefore locks the HNH and RuvC nuclease domains (glossary) in inactive conformations [50].…”

Section: Allosteric Inhibition and Clustering Of Effector Complexesmentioning

confidence: 99%

See 1 more Smart Citation

Diversity of molecular mechanisms used by anti-CRISPR proteins: the tip of an iceberg?

Hardouin

Goulet

2020

Biochemical Society Transactions

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Bacteriophages (phages) and their preys are engaged in an evolutionary arms race driving the co-adaptation of their attack and defense mechanisms. In this context, phages have evolved diverse anti-CRISPR proteins to evade the bacterial CRISPR–Cas immune system, and propagate. Anti-CRISPR proteins do not share much resemblance with each other and with proteins of known function, which raises intriguing questions particularly relating to their modes of action. In recent years, there have been many structure–function studies shedding light on different CRISPR–Cas inhibition strategies. As the anti-CRISPR field of research is rapidly growing, it is opportune to review the current knowledge on these proteins, with particular emphasis on the molecular strategies deployed to inactivate distinct steps of CRISPR–Cas immunity. Anti-CRISPR proteins can be orthosteric or allosteric inhibitors of CRISPR–Cas machineries, as well as enzymes that irreversibly modify CRISPR–Cas components. This repertoire of CRISPR–Cas inhibition mechanisms will likely expand in the future, providing fundamental knowledge on phage–bacteria interactions and offering great perspectives for the development of biotechnological tools to fine-tune CRISPR–Cas-based gene edition.

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Structures of Neisseria meningitidis Cas9 Complexes in Catalytically Poised and Anti-CRISPR-Inhibited States

Cited by 95 publications

References 56 publications

Biochemically diverse CRISPR-Cas9 orthologs

Biochemically diverse CRISPR-Cas9 orthologs

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

Diversity of molecular mechanisms used by anti-CRISPR proteins: the tip of an iceberg?

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