Fig. 7: Structural elements involved in dsDNA binding by the Csy-AcrIF24 complex.
a EMSA used to test the non-specific DNA binding ability of Csy-AcrIF24 with mutations in the PAM recognition loop of Cas8f using dsDNASP and dsDNANS. Reactions were performed as in Fig. 5c. Please see the right panels of Figs. 5c, d for WT controls of the experiments using dsDNASP and dsDNANS, respectively. b EMSA used to test the induction of non-specific DNA binding by AcrIF24 or its mutants using dsDNASP and dsDNANS. Reactions were performed as in Fig. 5a. c EMSA used to test the non-specific DNA binding ability of Csy-AcrIF24 with mutations both in AcrIF24 and Csy using dsDNASP and dsDNANS. Reactions were performed as in Fig. 5c. Please see the right panels of Figs. 5c, d for WT controls of the experiments using dsDNASP and dsDNANS, respectively. d Mutations impairing the induction of non-specific DNA binding of AcrIF24 decreased its capacity of inhibition in the in vitro cleavage activity assay. Reactions were performed as in Fig. 1a except that the concentrations of AcrIF24 or its mutants were set as 0.16 μM. Data are presented as mean values ± SD; n = 3. Two-sided t test was performed. The “*“ indicates that there is a significant difference between the quantitative data (P < 0.05). *P = 0.0098, 0.0125 (left to right). n.s, not significant, P = 0.7685. e Phage plaque assay verified that mutations impairing the induction of non-specific DNA binding of AcrIF24 decreased its capacity of inhibition of the type I-F system in vivo. Data are presented as mean values ± SD; n = 3. A two-sided t test was performed. The “*“ indicates that there is a significant difference between the quantitative data (P < 0.05). *P = 0.0003, 0.0015, 0.0006, 0.0005 (left to right). f Schematic illustration of working mechanisms of AcrIF24 on the inhibition of type I-F CRISPR-Cas system. Direct interaction with Csy and induction of its non-specific DNA binding both contribute to the inhibition by AcrIF24.
