Figure 3: AcrF1 in vitro binding strength correlates with in vivo activity.

(a) EMSA was used to assay binding of the Csy complex to target DNA in the presence of wild-type or mutant AcrF1 proteins. The anti-CRISPR proteins were added to the Csy complex in 10-, 100- or 1,000-fold excess. The ‘DNA+CC’ lanes contain DNA and Csy complex with no AcrF1. (b) Mixtures of Csy complex and AcrF1wild type or Y6A mutant were fractionated by size exclusion chromatography. The lanes show (1) peak fractions for the Csy complex with or without AcrF1 bound and (2) peak fractions for unbound AcrF1. (c) The indicated AcrF1-FLAG mutant proteins were pre-mixed with the Csy complex, and then competed by addition of wild-type AcrF1-HA. Csy complexes were isolated by size exclusion chromatography and the peak fraction from each sample was analysed by SDS–PAGE followed by silver staining. Western blots of the same gel, shown below, were used to identify the differentially tagged AcrF1 proteins. (d) Wild-type AcrF1-FLAG was incubated with Csy complex and then competed with wild-type AcrF1-3xHA for 16 h. Size exclusion chromatography was used to separate the AcrF1 bound to the Csy complex (1) from free AcrF1 (2).