PMID:28041849

Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.

Four type II-A Cas9 inhibitors are encoded by Listeria monocytogenes prophages

"This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages."
AcrIIA2 and AcrIIA4 inhibit the heterologous S. pyogenes Cas9 in bacteria and in human cells

"Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells."
Prophage-encoded Cas9 inhibition is widespread in the species rather than exceptional

"More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation."
Breadth of inhibition is attributed to a surface conserved between Cas9 orthologues

"AcrIIA2 and AcrIIA4 likely target regions conserved between the two Cas9 proteins"
The AcrIIA4 reference sequence used in this work is GenBank AEO04689.1

"AcrIIA2- (AEO04363.1), AcrIIA3- (CBY03209.1) and AcrIIA4- (AEO04689.1) homologous protein sequences were acquired by BLASTp searches"

PMID:28602637

Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9.

AcrIIA4 competitively occupies both the PAM-recognition surface and the RuvC catalytic pocket

"demonstrate that AcrIIA4 competitively occupies both PAM-interacting and non-target DNA strand cleavage catalytic pockets"
The inhibitor targets the residues essential for PAM duplex recognition and blocks DNA access to the RuvC catalytic residues

"AcrIIA4 preferentially targets critical residues essential for PAM duplex recognition, as well as blocks target DNA access to key catalytic residues lining the RuvC pocket"
AcrIIA4 buries roughly three times the surface that the PAM duplex itself buries, which is why it outcompetes the duplex

"AcrIIA4 targets the concave surface formed by the Topo, CTD and RuvC domains in the SpyCas9-sgRNA binary complex and buries an area of 1,693 Å2 on ternary complex formation (Figure 4A). By contrast, positioning of the PAM duplex of dsDNA on the surface formed by Topo and CTD domains of the SpyCas9-sgRNA binary complex buries an area of 553 Å"
A protruding loop of AcrIIA4 enters the RuvC active site

"the protruding β1–β2 loop of AcrIIA4 inserts into the active site of RuvC domain"
Binding is selective for the guide-RNA-loaded form of Cas9

"Selective Binding of AcrIIA4 to sgRNA-bound SpyCas9"

PMID:28448066

Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR protein.

AcrIIA4 structurally mimics the PAM to occupy the PAM-interacting site and block double-stranded DNA recognition

"AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9."
A second, independent inhibitory contact shields the RuvC active site

"AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by shielding its RuvC active site."
Interaction with Cas9 requires the guide RNA

"Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner."
The AcrIIA4 binding site on Cas9 is created by guide RNA binding

"Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding."

PMID:29497118

Solution structure and dynamics of anti-CRISPR AcrIIA4, the Cas9 inhibitor.

AcrIIA4 is a strongly acidic protein with negative charge concentrated in the Cas9-contacting loops

"AcrIIA4 is an acidic protein (pI ~4.2), and the surface electrostatic potential indicates that negative charges are densely populated in the β3–α2 loop, the α2–α3 loop and the beginning of the α3 helix"
The acidic patches are interpreted as mimicking nucleic acid phosphate groups at the PAM interaction site

"It has been suggested that the negatively charged patches mimic the phosphate groups of nucleic acids that associate with the PAM interaction site of SpyCas9"
The same mimicry strategy is used by the sequence-unrelated type I-F inhibitor AcrF2

"which was also found in AcrF2 proteins inhibiting the subtype I-F effector complex"

PMID:28844692

A Broad-Spectrum Inhibitor of CRISPR-Cas9.

AcrIIA4 is described in the primary literature as a DNA mimetic that prevents DNA binding, in explicit contrast to the HNH-blocking inhibitors

"Both of these mechanisms are different from that of the anti-CRISPR protein AcrIIA4, which acts as a DNA mimetic that prevents DNA binding by occupying t"

PMID:28706995

Disabling Cas9 by an anti-CRISPR DNA mimic.