AcrIIC2 was discovered via a guilt-by association (GBA) approach, which was based on the strong co-occurrence and clustering of acr and anti-CRISPR associated (aca) genes through proximity and homology searches. Subsequent structural and biochemical work has shown that the AcrIIC2 dimer inhibits Cas9 complex assembly through interactions with the Cas9 positively charged bridge helix, that prevents crRNA binding and DNA loading into Cas9. Type IIC CRISPR-Cas systems are the simplest of the type II systems, employing only three Cas proteins, Cas9, Cas1, and Cas2. Almost all type II Acr proteins characterized to date directly interact with the Cas9 endonuclease, although by distinct mechanisms. The type II CRISPR/Cas subtype has a distinct crRNA-guided surveillance complex encoded by cas9 (formerly csn1), cas1, cas2, and csn2 (for type IIA) or cas4 (for type IIB) genes, all located in a single transcriptional unit directly upstream of the CRISPR locus. Cleavage of the DNA target in type II systems is carried out by Cas9 which is an RNA-guided double-stranded DNase with two independent nuclease domains, HNH and RuvC. Due to the reliance of the type II system on a single protein for function, Cas9 homologs derived from different subtypes and species have been utilized for numerous gene editing applications. AcrIIC1 and AcrIIC2 can target a broad spectrum of Cas9 orthologs from different subtypes which is important for designing AcrIIC1- and AcrIIC2-based molecular tools for biotechnological applications. CRISPR-Cas immune systems are used by certain prokaryotes and archaea to resist the invasion of foreign nucleic acids such as phages or plasmids. Anti-CRISPRs are small proteins which are the natural inhibitors for CRISPR-Cas systems; encoded on bacterial and archaeal viruses, they allow the virus to evade host CRISPR-Cas systems. The CRISPR-Cas-mediated adaptive immune response can be divided into three steps, including the acquisition of spacer derived from invading nucleic acids, crRNA processing, and target degradation. Theoretically, Acr proteins could suppress any step to disrupt the CRISPR-Cas system. Acr proteins are diverse with no common sequence or structural motif, and they inhibit a wide range of CRISPR-Cas systems with various inhibition mechanisms. CRISPR-Cas systems are divided into two classes (1 and 2) and six types (class 1: types I, III and IV; class 2: types II, V and VI). Class 2 systems employ a single multi-domain effector Cas9 protein complex that performs target recognition and cleavage. Acr families are named for their type and subtype which are numbered sequentially as they are discovered.
Feature 1:Cas9 binding site [polypeptide binding site]
Evidence:
Structure:6JDJ: Bacteriophage AcrIIC2 homodimer binds partial Neisseria meningitidis Cas9 (which includes the arginine-rich bridge helix); contacts at 4A
Comment:The Cas9 arginine-rich bridge helix, which connects the REC and NUC lobes, is a universal feature of Cas9 proteins that is targeted by homodimerized AcrIIC2 to inhibit loading of the guide RNA molecule, thereby preventing formation of the active CRISPR-Cas9 surveillance complex.
Jiyao W et al.(2023). "The conserved domain database in 2023.",