Disabling Cas9 by an anti-CRISPR DNA mimic

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Microbial Immunology

Background:

• CRISPR-Cas9 gene editing is derived from microbial adaptive immunity, targeting bacteriophages.
• Natural CRISPR-Cas9 inhibitors (anti-CRISPR proteins) are known but their mechanisms and applications in mammalian cells are unclear.
• Understanding these inhibitors is crucial for controlling Cas9-mediated gene editing.

Purpose of the Study:

• To elucidate the mechanism of action for the anti-CRISPR protein AcrIIA4.
• To investigate the potential of AcrIIA4 in modulating Cas9-mediated gene editing in human cells.

Main Methods:

• Cryo-electron microscopy was used to determine the structure of the Cas9-sgRNA-AcrIIA4 complex at 3.9 Å resolution.
• Binding assays and order-of-addition experiments were performed to assess AcrIIA4's interaction with Cas9, sgRNA, and DNA.
• Timed delivery of AcrIIA4 (protein or plasmid) into human cells to evaluate its effect on gene editing outcomes.

Main Results:

• AcrIIA4 binds specifically to assembled Cas9-sgRNA complexes, not Cas9 alone, with 1:1 stoichiometry.
• Structural analysis revealed AcrIIA4 binds to the Cas9 region that recognizes the DNA protospacer adjacent motif, inhibiting DNA binding.
• Timed delivery of AcrIIA4 in human cells resulted in controlled on-target gene editing and reduced off-target mutations.

Conclusions:

• AcrIIA4 functions by sterically hindering Cas9-sgRNA binding to target DNA.
• Anti-CRISPR proteins like AcrIIA4 can be mechanistically understood and utilized to precisely control CRISPR-Cas9 gene editing.
• These findings establish a basis for using inhibitors to modulate gene editing efficiency and specificity in mammalian systems.