Supplementary Figure 2: Biochemical characterization of dsDNA cleavage by FokI-EcoCascade.

a, Design of the target plasmid, in which λ1 and λ2 target sites flanked interspacer DNA in a “PAM-in” configuration. b, Plasmid DNA cleavage as a function of ionic strength. The target plasmid from a, or a non-target plasmid lacking the λ1 and λ2 target sites, was incubated with dimeric FokI-EcoCascade complexes for 30 minutes at 37 °C in a reaction buffer containing NaCl at either 50, 100, 150, or 200 mM. At low salt concentrations, both non-target and target plasmid DNA substrates are non-specifically nicked and degraded, as evidenced by the appearance of low-molecular weight DNA at the bottom of the gel (Deg, degraded DNA). At higher salt concentrations, the non-target plasmid remains intact in its supercoiled state, whereas the target plasmid is linearized upon DSB formation. FokI-Cas8 in these reactions contained a 30-aa linker. c, Plasmid DNA cleavage as a function of interspacer and FokI-Cas8 linker length. The non-target plasmid or target plasmids with interspacers ranging from 20–50-bp were incubated with FokI-EcoCascade containing FokI-Cas8 linker lengths of 8, 10, 12, 15, 16, or 30 aa. The 30-bp interspacer substrate is primarily linearized, whereas shorter or longer interspacer substrates are efficiently nicked but not converted into linear form. The longest 30-aa linker length is still able to produce linearized DNA for longer interspacer substrates, and was thus selected for subsequent experiments. d, DNA DSBs require the presence of Cas8-less Cascade and FokI-Cas8, both paired gRNAs, and both target half-sites. Control reactions, which contained neither target site or only one half-site, or only one of the two paired gRNAs, failed to yield a linearized DNA product. The target plasmid was efficiently cleaved either by co-incubation with FokI-Cas8 and two separately purified Cas8-less Cascade subcomplexes containing either gRNA (lane 10), or by FokI-Cas8 and a single Cas8-less Cascade subcomplex purified from an expression strain encoding both paired gRNAs (λ1+λ2; lane 11). e, The target plasmid is no longer cleaved when both half-sites contain mutations in either the PAM or the seed sequence. f, Target plasmid cleavage as a function of FokI-EcoCascade concentration, and comparison to Cas9-sgRNA. 150 ng plasmid DNA was incubated with protein-RNA complexes ranging from ~3–100 nM for 30 minutes at 37 °C, prior to agarose gel electrophoresis. g, Target plasmid cleavage as a function of incubation time. 200 ng plasmid DNA was incubated with 200 nM FokI-EcoCascade or Cas9-sgRNA complexes at 37 °C from 0–30 minutes, prior to agarose gel electrophoresis. N, nicked; L, linear; SC, supercoiled. All experiments in this panel were repeated two independent times with similar results; uncropped gels can be found in Source Data Fig. 2.