Interaction between AcrIF23 and Cas2/3 is essential for its inhibition activity.A, AcrIF23 inhibits in vitro ssDNA cleavage by Cas2/3. The reaction system contains 200 nM Cas2/3, 25 nM ssDNA, and 0.5, 1, or 2 μM AcrIF23. B, AcrIF23 is shown in cartoon and surface model with views of its two sides. Mutated residues are shown in sticks. Residues whose mutations markedly reduce the inhibition of the ssDNA cleavage are marked with red texts. C and D, inhibition of in vitro ssDNA cleavage of the 20 AcrIF23 mutants. The experiment was performed as in A. E, MST assay of the binding between Cas2/3 and AcrIF23 and its mutants. Error bars indicate the s.d. of three independent measurements. Fitted binding curve is shown for AcrIF23-Cas2/3 binding.