Figure 2.

Bacterial directed evolution system for selecting endonuclease variants with high specificity. (a) Schematics of the plasmids used in the bacterial selection system. The pENDO-HE plasmid expresses the endonuclease and contains an HE target for negative selection. The pCcdB plasmid expresses the CcdB protein, a DNA gyrase poison, which causes bacterial cell death if the target sites on the plasmid are not cleaved by the endonuclease. Both the modifications to allow for specificity selection and increase system throughput are shown in red. (b) Comparisons of endonuclease activity with kinetic data from in vitro cleavage assays, survival in the original selection system and survival in the improved system with the specificity selection component. An extended comparison of survival and in vitro cleavage activity for multiple single base-pair substitutions in the I-AniI target is available in Supplementary Figure S1.