(A) Schematic showing kinetic and equilibrium parameters inserted into the KinTek Global Kinetic Explorer software for modelling the type III CRISPR defence illustrated in Figure 1. Parameters have been determined in this study with the following exceptions: Crn1 rate constant of 0.46 min−1 at 70°C estimated from rate of 0.23 min−1 measured at 60°C and AcrIII-1 rate constant of 21.6 min−1 at 70°C estimated from rate of 5.4 min−1 measured at 50°C (Athukoralage et al., 2020). The parameter ‘target-Csm-ATP’ was set at 6, 60 or 600 µM in simulations. Underscores connecting two variables indicate their relationship in a complex. cA4, cyclic tetra-adenylate; Crn1, CRISPR ring nuclease 1; AcrIII-1, viral ring nuclease anti-CRISPR SIRV1 gp29; Csx1, CRISPR ancillary ribonuclease; A2 >P, di-adenylate containing 2’,3’ cyclic phosphate (product of cA4 cleavage). (B) Free cA4 (600 µM, blue; 60 µM, red; 6 µM, green) concentration and (C), RNA cleavage in presence of 1 µM Crn1 and 1 µM Csx1. (D) and (E) show equivalent plots in the presence of 1 µM AcrIII-1. Insets where present show a magnified view of the start of each plot. (F) 3D plot visualising concentration of RNA (1000 µM at start) cleaved by Csx1 in response to 60 µM cA4 made by Csm complex, 1 µM Crn1 and varying amounts of AcrIII-1 across a range of doubling endpoints. (G) 3D plot visualising concentration of RNA (1000 µM at start) cleaved by Csx1 in response to 60 µM cA4 made by Csm complex, and varying concentrations of Csx1, Crn1 and AcrIII-1 enzymes.