The CRISPR-Cas system serves as an adaptive immune defense in bacteria and archaea, targeting and degrading foreign nucleic acids. Bacteriophages, in turn, have evolved countermeasures known as anti-CRISPR (Acr) proteins that neutralize CRISPR-Cas activity.
These proteins exhibit diverse mechanisms of inhibition, such as preventing DNA/RNA binding, disrupting complex assembly, or promoting degradation via host proteases. Among these inhibitors, AcrIE7 stands out for targeting the type I-E CRISPR-Cas system, although its mechanism of action remained poorly understood until this study.
This study focused on characterizing how AcrIE7 inhibits the CRISPR-Cas system. The researchers discovered that AcrIE7 blocks the function of Cas3, a critical DNA-degrading enzyme in type I-E systems, by binding to single-stranded DNA (ssDNA) within the R-loop structure that forms during target recognition. This represents a distinct mode of inhibition compared to previously characterized Acr proteins that target other parts of the CRISPR machinery.
Unique AcrIE7 Structure Revealed, with Specific ssDNA Binding via Basic Surface Patches
Using crystallography, the structure of AcrIE7 was solved and found to adopt a seven-helix bundle fold. Notably, this structure deviated significantly from predictions made by AlphaFold, suggesting a unique conformation not commonly seen among Acr proteins. Additional structural comparisons through DALI analysis revealed only weak similarity to known proteins, reinforcing the uniqueness of AcrIE7’s fold and function.
CRISPR Inhibitor AcrIE7 Blocks DNA Degradation by Binding R-Loop ssDNA with a Unique Structural FoldElectrostatic surface analysis identified two basic patches likely responsible for nucleic acid binding. Experimental assays confirmed that AcrIE7 selectively binds ssDNA rather than double-stranded DNA. Binding led to the formation of distinct protein-DNA complexes, and further size analysis confirmed AcrIE7 operates as a monomer. Mutational studies pinpointed specific residues (K6 and R49) as essential for ssDNA interaction, indicating the functional significance of the first basic patch.
AcrIE7 Inhibits Cas3 by Binding R-Loop ssDNA in CRISPR Systems
To determine whether AcrIE7’s ssDNA binding inhibits Cas3, the authors conducted cleavage assays using fluorescently labeled ssDNA. The results showed that AcrIE7 binding protected the ssDNA from Cas3-mediated degradation. Further testing with mutant versions of AcrIE7 that had reduced binding capability demonstrated diminished inhibitory effects, confirming the importance of direct ssDNA binding for AcrIE7’s function.
The study extended its investigation to a complete CRISPR-Cas system including Cascade and Cas3. When a labeled DNA duplex was introduced, Cascade-mediated R-loop formation exposed ssDNA, which became a target for Cas3 cleavage. AcrIE7 successfully inhibited this cleavage by binding the exposed ssDNA, and again, mutants with weaker binding affinity were less effective. This reinforced the conclusion that AcrIE7 acts by targeting ssDNA at the R-loop, rather than interacting directly with Cas proteins like Cas3 or Cascade.
This study introduces a novel CRISPR-Cas inhibition mechanism whereby AcrIE7 binds to R-loop-exposed ssDNA to block Cas3 cleavage, a strategy distinct from previously described anti-CRISPR methods. The specificity of this inhibition to type I-E and I-C systems suggests a possible preference for particular Cascade-generated R-loops. Although the study stops short of proving direct interaction within the Cascade-formed R-loop, it lays the groundwork for future research to confirm these interactions and better understand the evolutionary significance of AcrIE7’s unique strategy.
