Figure 4.
Binding affinity of Cas9 proteins for sgRNA and DNA. (A). Binding affinity of Cas9 proteins for cognate and non-cognate guides as determined by filter binding assays. Measurements were made in at least triplicate to determine KD and a representative replicate is shown. Data were fit to a binding isotherm. (KD ± SD for Spy Cas9 to Spy sgRNA, 28 pM ± 6 pM; Spy Cas9 to Cdi sgRNA, 146 ± 11nM; Cdi Cas9 to Spy sgRNA, 115 ± 51nM; Cdi Cas9 to Cdi sgRNA, 0.56 ± 0.13nM) (B). Binding affinity of Cas9 proteins for dsDNA, target ssDNA and non-target ssDNA as measured by electrophoretic mobility shift assay (EMSA). Each Cas9 protein was incubated with its cognate guide. Measurements were made in at least triplicate to determine KD and a representative replicate is shown. Data were fit to a binding isotherm. (KD ± SD [where appropriate] for Spy Cas9 to dsDNA, 25 ± 11 nM; Spy Cas9 to target ssDNA, 40 ± 11 nM; Spy Cas9 to non-target ssDNA, >1000 nM; Cdi Cas9 to dsDNA, >1000 nM; Cdi Cas9 to target ssDNA, 58 ± 32 nM; Cdi Cas9 for non-target ssDNA, >1000 nM. See also Figure S5 and Table S2