AcrIF23 does not prevent Cas2/3 recruitment by the Csy-DNA complex.A, comparison of the inhibition capacity of ssDNA cleavage by Cas2/3 between AcrIF23 and AcrIF3. The reaction system contains 200 nM Cas2/3, 25 nM ssDNA, and 0.5, 1, or 2 μM AcrIF23 or AcrIF3. B, AcrIF3 but not AcrIF23 prevents Cas2/3 recruitment by the Csy-DNA complex. Reactions were performed with 1.6 μM Csy complex, 0.1 μM dsDNA, 1 μM Cas2/3 and 0.5, 1, or 2 μM AcrIF23 or AcrIF3. C, EMSA experiments to test the effect on ssDNA binding of Cas2/3 by AcrIF23 and AcrIF3. The reaction system contains 1.5 μM Cas2/3, 100 nM ssDNA, and 0.03125, 0.125, 0.5 or 2 μM AcrIF23 or AcrIF3. In the last lanes of the two panels, AcrIF3 or AcrIF23 was added at 2 μM. MST, microscale thermophoresis; ssDNA, single-stranded DNA.