AcrIF23 functions after the hybridization between the Csy complex and target DNA.A, AcrIF23 inhibits the DNA cleavage regardless of the incubation order. The reaction system contains 320 nM Csy complex, 50 nM dsDNA, and 0.5, 1, or 2 μM AcrIF23. The Csy complex is incubated first with AcrIF23 or DNA, respectively. After incubation of the Csy/DNA/AcrIF23, Cas2/3 was added with a final concentration of 200 nM and incubated for 20 min at 37 °C. B, EMSA to test the inhibition of Csy-DNA binding by AcrIF23. The reaction system contains 1.6 μM Csy complex, 0.1 μM dsDNA, and 0.5, 1, or 2 μM AcrIF23. Similarly, the Csy complex is incubated first with AcrIF23 or DNA, respectively. dsDNA, double-stranded DNA; EMSA, electrophoretic mobility shift assay.