Insights into the dual functions of AcrIF14 during the inhibition of type I-F CRISPR–Cas surveillance complex

Abstract

CRISPR–Cas systems are bacterial adaptive immune systems, and phages counteract these systems using many approaches such as producing anti-CRISPR (Acr) proteins. Here, we report the structures of both AcrIF14 and its complex with the crRNA-guided surveillance (Csy) complex. Our study demonstrates that apart from interacting with the Csy complex to block the hybridization of target DNA to the crRNA, AcrIF14 also endows the Csy complex with the ability to interact with non-sequence-specific dsDNA as AcrIF9 does. Further structural studies of the Csy–AcrIF14–dsDNA complex and biochemical studies uncover that the PAM recognition loop of the Cas8f subunit of the Csy complex and electropositive patches within the N-terminal domain of AcrIF14 are essential for the non-sequence-specific dsDNA binding to the Csy–AcrIF14 complex, which is different from the mechanism of AcrIF9. Our findings highlight the prevalence of Acr-induced non-specific DNA binding and shed light on future studies into the mechanisms of such Acr proteins.

Anti-CRISPR proteins function through thermodynamic tuning and allosteric regulation of CRISPR RNA-guided surveillance complex

“…In Liu et al. , it was observed that AcrIF14 (which also interacts with Cas7.4f and Cas7.6f) induces non-specific dsDNA binding when complexed with Csy, though the mechanism of this binding differs from that of non-specific dsDNA binding to the AcrIF9–Csy complex ( 59 ).…”

Advances in rapid detection of Pseudomonas aeruginosa with DNase-based sensors

“…As DNase degrades the substrate, changes in the markers' current or potential are detected through electrochemical methods, effectively detecting Pseudomonas aeruginosa. Another method, the DNA enzyme-fluorescent dye approach, sees DNA enzyme binding to target DNA and catalyzing its degradation, leading to the dissociation of DNA bound with fluorescent dye, inducing changes in fluorescence signals [22]. Detecting shifts in fluorescence intensity promptly reveals the presence of Pseudomonas aeruginosa.…”

Structural and mechanistic insights into the inhibition of type I-F CRISPR-Cas system by anti-CRISPR protein AcrIF23

“…Specifically, AcrIF1/2/4/6/7/8/9/10/14 played their roles though direct interaction with the Csy complex ( 13 , 14 , 15 , 16 , 17 , 18 ), while AcrIF3 stably binds the Cas2/3 protein ( 19 , 20 , 21 ). Out of these, interestingly, other than sterically inhibiting the binding between the Csy complex and target DNA, AcrIF9, and AcrIF14 have also been found to mislead the Csy complex to absorb nonsequence–specific DNA, which has been demonstrated to contribute to the inhibition activity of the Acr proteins ( 18 , 22 , 23 ). Distinct from the above ten Acr proteins, AcrIF11 does not stably bind the Csy complex, instead, it has been reported to possess ADP-ribosylation activity on the N250 residue of the Cas8f subunit of the Csy complex, a key residue required for protospacer adjacent motif recognition ( 24 ).…”