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

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• AcrIIA15 is an anti-CRISPR protein known to inhibit Staphylococcus aureus Cas9 (SaCas9).
• Previous research indicated dual functions for AcrIIA15, but the structural and biochemical underpinnings were not fully understood.

Purpose of the Study:

• To elucidate the structural and biochemical basis of AcrIIA15's dual inhibitory functions against SaCas9.
• To understand the mechanism of AcrIIA15 in blocking SaCas9 activity and regulating its own transcription.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine the structure of AcrIIA15 complexed with SaCas9-sgRNA.
• X-ray crystallography to obtain structures of AcrIIA15 bound to promoter DNA.

Main Results:

• The C-terminal domain (CTD) of AcrIIA15 mimics double-stranded DNA (dsDNA), blocking the protospacer adjacent motif (PAM) recognition site of SaCas9.
• The N-terminal domain (NTD) of AcrIIA15 facilitates dimerization and recognition of dsDNA.
• AcrIIA15 forms a supercomplex by simultaneously binding SaCas9-sgRNA and promoter DNA, involving two Cas9s, two CTDs, and an NTD dimer bound to dsDNA.

Conclusions:

• Structural and biochemical data reveal AcrIIA15's dual mechanism: CTD inhibits SaCas9 via PAM mimicry, and NTD mediates DNA binding for transcriptional autoregulation.
• The formation of a supercomplex highlights a novel mode of interaction between anti-CRISPR proteins, Cas9, and host DNA.
• Findings advance the understanding of CRISPR-Cas systems, phage-host dynamics, and transcriptional regulation by anti-CRISPR proteins.