Structural basis of AcrIF24 as an anti-CRISPR protein and transcriptional suppressor

Mukherjee, Indranil Arun; Gabel, C.; Noinaj, Nicholas; Bondy‐Denomy, Joseph; Chang, Leifu

doi:10.1038/s41589-022-01137-w

Cited by 8 publications

(3 citation statements)

References 41 publications

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“…These findings suggest that dimerization is essential for the recognition of dsDNA by AcrIIA15, consistent with the structural observation that the HTH domains of a dimer bind to the inverted-repeat sequence of the DNA. Our results align well with a previous study demonstrating that the HTH dimer of AcrIF24 interacts with DNA 29 .…”

Section: Resultssupporting

confidence: 93%

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An anti-CRISPR that represses its own transcription while blocking Cas9-target DNA binding

Deng,

Sun,

et al. 2024

Nat Commun

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AcrIIA15 is an anti-CRISPR (Acr) protein that inhibits Staphylococcus aureus Cas9 (SaCas9). Although previous studies suggested it has dual functions, the structural and biochemical basis for its two activities remains unclear. Here, we determined the cryo-EM structure of AcrIIA15 in complex with SaCas9-sgRNA to reveal the inhibitory mechanism of the Acr’s C-terminal domain (CTD) in mimicking dsDNA to block protospacer adjacent motif (PAM) recognition. For the N-terminal domain (NTD), our crystal structures of the AcrIIA15-promoter DNA show that AcrIIA15 dimerizes through its NTD to recognize double-stranded (ds) DNA. Further, AcrIIA15 can simultaneously bind to both SaCas9-sgRNA and promoter DNA, creating a supercomplex of two Cas9s bound to two CTDs converging on a dimer of the NTD bound to a dsDNA. These findings shed light on AcrIIA15’s inhibitory mechanisms and its autoregulation of transcription, enhancing our understanding of phage-host interactions and CRISPR defense.

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Section: Resultssupporting

confidence: 93%

“…The HTH domains for all known fused type II anti-CRISPRs are located at the NTD, whereas type I-F anti-CRISPR AcrIF24 has a C-terminal HTH domain 29 . Both AcrIIA15 and AcrIF24 can form dimers and bind to the Acr promoter DNA to repress its transcription.…”

Section: Discussionmentioning

confidence: 99%

An anti-CRISPR that represses its own transcription while blocking Cas9-target DNA binding

Deng,

Sun,

et al. 2024

Nat Commun

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“…7a and 7b). The presence of the HTH domain suggests the potential of AcrIF25 to act as an Aca protein, reminiscent of the Acr-Aca protein AcrIF24 28,29 . We attempted to determine the crystal structure of AcrIF25, but possibly due to the high flexibility of the loop connecting CTD and NTD, we were only successful in solving the structure of the HTH NTD (Supplementary Data.…”

Section: Both Acrif25 and Acrif26 Induce The Non-specific Dna Binding...mentioning

confidence: 99%

CRISPR-repressed toxin-antitoxin provides population-level immunity against diverse anti-CRISPR elements

Shu,

Wang,

et al. 2024

Preprint

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Prokaryotic CRISPR-Cas systems are highly vulnerable to phage-encoded anti-CRISPR (Acr) factors. How CRISPR-Cas systems protect themselves remains unclear. Here, we uncovered a broad-spectrum anti-anti-CRISPR strategy involving a phage-derived toxic protein. Transcription of this toxin is normally reppressed by the CRISPR-Cas effector, but is activated to halt cell division when the effector is inhibited by any anti-CRISPR proteins or RNAs. We showed that this abortive infection-like effect efficiently expels Acr elements from bacterial population. Furthermore, we exploited this anti-anti-CRISPR mechanism to develop a screening method for specific Acr candidates for a CRISPR-Cas system, and successfully identified two distinct Acr proteins that enhance the binding of CRISPR effector to non-target DNA. Our data highlight the broad-spectrum role of CRISPR-repressed toxins in counteracting various types of Acr factors, which illuminates that the regulatory function of CRISPR-Cas confers host cells herd immunity against Acr-encoding genetic invaders, no matter they are CRISPR-targeted or not.

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Anti-CRISPR Discovery: Using Magnets to Find Needles in Haystacks

Forsberg

2023

Journal of Molecular Biology

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Structural basis of AcrIF24 as an anti-CRISPR protein and transcriptional suppressor

Cited by 8 publications

References 41 publications

An anti-CRISPR that represses its own transcription while blocking Cas9-target DNA binding

An anti-CRISPR that represses its own transcription while blocking Cas9-target DNA binding

CRISPR-repressed toxin-antitoxin provides population-level immunity against diverse anti-CRISPR elements

Anti-CRISPR Discovery: Using Magnets to Find Needles in Haystacks

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