Bondy-Denomy et al. purified AcrF1, AcrF2 and AcrF3, which they had previously identified as inhibitors of the type I-F CRISPR–Cas system in Pseudomonas aeruginosa. In this system, the Csy complex (comprising Csy1, Csy2, Csy4 and six copies of Csy3, together with a CRISPR RNA (crRNA)) recognizes invading DNA, which is then degraded by the Cas3 nuclease. Using size-exclusion chromatography (SEC), the authors showed that AcrF1 and AcrF2 both interact with the Csy complex, whereas AcrF3 interacts with Cas3. Furthermore, electrophoretic mobility shift assays showed that AcrF1 and AcrF2 block the binding of the Csy complex to the target DNA, whereas AcrF3 blocks the binding of Cas3 to the target-bound Csy complex.
To see whether these in vitro findings were representative of anti-CRISPR activity in vivo, the authors studied host–phage interactions in P. aeruginosa. First, they showed that overexpression of Csy components — to dilute interactions with anti-CRISPR proteins — reduced the viability of phage encoding AcrF1 or AcrF2, but not AcrF3, which confirmed that the Csy complex has direct roles in the anti-CRISPR mechanisms of AcrF1 and AcrF2. Next, the role of Cas3 was examined by exploiting a phenomenon in which the loss of Cas3 activity converts the Csy complex into a transcriptional repressor, owing to the stable physical interaction with target DNA that is formed in the absence of degradative activity. To monitor transcriptional repression resulting from Cas3 inhibition, the authors used an endogenous reporter gene (phzM) that is required for the production of the blue-green pigment pyocyanin. phzM was transcriptionally repressed in the presence of AcrF3, but not AcrF1 or AcrF2, which confirmed the in vitro data supporting a direct interaction between AcrF3 and Cas3.
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