PMID:28785032

An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9.

Phage D4276 escapes Streptococcus thermophilus CRISPR immunity more than 40,000-fold better than comparable phages

"we found one that evaded the CRISPR-encoded immunity >40,000× more often than the others"
A single cloned gene of phage D4276 is wholly responsible for the loss of CRISPR immunity

"Through systematic cloning of its genes, we identified an Acr solely responsible for the abolished immunity."
The protein also abolishes immunity conferred by the heterologous Streptococcus pyogenes Cas9 in a different bacterial genus

"We extended our findings by demonstrating activity in another S. thermophilus strain, against unrelated phages, and in another bacterial genus immunized using the heterologous SpCas9 system favoured for genome editing."
This is the first anti-CRISPR found in a virulent rather than temperate phage or prophage

"we have identified an unrelated Acr in a virulent phage of Streptococcus thermophilus"

PMID:30046034

Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins.

AcrIIA5 orthologues including the D4276 protein fully restore plaquing of phages targeted by the St1Cas9 (CR1) system

"All AcrIIA6 and AcrIIA5 orthologs completely restored the plaquing efficiency of phages targeted by the CR1 system of S. thermophilus (Fig. 4), indicating they are blocking the activity of St1Cas9."
AcrIIA5 almost completely abolishes plasmid-borne SpCas9 activity in Lactococcus lactis

"Both AcrIIA5s almost completely abolished SpCas9 activity in this bacterial system"
The D4276 orthologue specifically is only weakly active against St3Cas9, unlike the 87% identical D1126 orthologue

"Notably, AcrIIA5D4276 comes from a phage infecting a S. thermophilus host which lacks the CR3 locus, and its activity is limited to a 10-fold inhibition of CR3 activity25."
AcrIIA5 has the broadest activity of all anti-CRISPRs tested in this study, in bacteria and in human cells

"Of all the Acrs tested in human cells and in the bacterial assays, AcrIIA5 displayed the broadest range of activity"
AcrIIA5 inhibits Cas9-mediated genome editing in human cells

"we demonstrate that both Acr families from S. thermophilus phages inhibit Cas9-mediated genome editing of human cells"

PMID:32544231

Intrinsic disorder is essential for Cas9 inhibition of anti-CRISPR AcrIIA5.

AcrIIA5 adopts a novel fold with an N-terminal intrinsically disordered region

"AcrIIA5 comprises seven β-strands and two α-helices, preceded by an extended N-terminal disordered region"
No structural homologue of the AcrIIA5 fold exists in the PDB

"A search for structural homologs of AcrIIA5 using the DALI program did not return a similar fold, indicating that AcrIIA5 adopts a novel fold"
Purified AcrIIA5 alone is sufficient to block Cas9-sgRNA cleavage of a double-stranded DNA target in vitro

"Purified AcrIIA5 completely inhibited target double-stranded DNA (dsDNA) cleavage of Cas9–sgRNA in the in vitro nuclease activity assay, demonstrating that AcrIIA5 alone is sufficient for Cas9 inhibition"
AcrIIA5 binds the assembled Cas9-sgRNA ribonucleoprotein directly, forming a ternary complex

"Titration of AcrIIA5 into Cas9–sgRNA resulted in sgRNA super-shifts owing to the ternary complex formation, demonstrating a direct interaction between AcrIIA5 and Cas9–sgRNA"
AcrIIA5 binds only guide-loaded Cas9 and not apo-Cas9

"We note that AcrIIA5 interacted only with the preformed Cas9–sgRNA complex, and not with apo-Cas9."
AcrIIA5 inhibits without competing with target-DNA binding, unlike the DNA-mimic inhibitors

"AcrIIA5 selectively binds to sgRNA-loaded Cas9 and inhibits the nuclease activity without competing with target DNA binding, which is unique among Cas9 inhibitors."
AcrIIA5 has no intrinsic ribonuclease activity on free or Cas9-bound sgRNA

"Treatment of free sgRNA or the Cas9–sgRNA complex with AcrIIA5 did not digest sgRNA, and the AcrIIA5-treated sgRNA bands remained intact in the urea gel"
The N-terminal disordered region is required for inhibition, supplying both the length needed for association and the positive charges needed to block activity

"the length of the IDR provides a scaffold for association between AcrIIA5 and Cas9–sgRNA, while the positive charges of the IDR serve as a regulatory switch that hinders Cas9 function upon binding"
A mobile beta3-beta4 loop in the folded core carries additional residues required for inhibition

"the key residues of AcrIIA5 required for Cas9 inhibition are located at the mobile loop region in the middle of the compact fold, in addition to the N-terminal IDR"
Inhibition is selective for Cas9 and does not extend to the type V-A nuclease Cas12a

"AcrIIA5 did not inhibit L. bacterium Cas12a, another RNA-guided endonuclease from the type V-A CRISPR–Cas system"

PMID:31722192

Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing.

AcrIIA5 from phage D4276 has the broadest Cas9 inhibitory spectrum described, covering all Cas9 proteins in common genome-editing use

"We discovered that the previously identifed anti-CRISPR, AcrIIA5 from Streptococcus thermophilus phage D4276 (Hynes et al., 2017), has the broadest Cas9 inhibitory activity described to date, inhibiting all of the Cas9 proteins commonly used in genome-editing applications."
AcrIIA5 potently inhibits nine diverse type II-A and type II-C Cas9 homologues

"here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing"
AcrIIA5 activity is accompanied by partial in vivo cleavage of the single-guide RNA, which the authors read as evidence of an RNA-directed mechanism

"We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction."
AcrIIA5 could not be purified in a soluble active form in this study, which is why its mechanism was approached indirectly

"Despite extensive efforts, AcrIIA5 could not be purified in a soluble and active form on its own."

PMID:31775029

AcrIIA5 Inhibits a Broad Range of Cas9 Orthologs by Preventing DNA Target Cleavage.

AcrIIA5 prevents target-DNA cleavage by Cas9 while leaving target-DNA binding intact

"AcrIIA5 inhibits Cas9 by preventing DNA target cleavage, but DNA target binding of Cas9 is unaffected."
The inhibited activity is that of the RuvC nuclease domain, independently of the HNH domain

"it can affect the activity of the RuvC nuclease domain of Cas9 independent of the HNH nuclease domain"
Inhibition covers type II-A, II-B and II-C Cas9 orthologues

"We found that AcrIIA5 is a potent and broad-spectrum inhibitor of CRISPR-Cas9, which can inhibit diverse Cas9 orthologs of type II-A, type II-B, and type II-C."