Directed Evolution of Improved Zinc Finger Methyltransferases
(A) Relative locations of the target site and non-target site on a plasmid linearized by NcoI digestion. For sequences of the target and non-target sites, refer to Figures 1C and D (B) The restriction endonuclease protection assay for methylation at the target and non-target site uses digestion with NcoI to linearize the plasmid and either FspI or SnaBI to assess the target and off-target methylation, respectively. FspI and SnaBI cannot digest a methylated site. Shown are results from select variants as well as the ‘wildtype’ heterodimeric enzyme (i.e. no mutations to residues 297–301) with or without a catalytically inactivating (C141S), or a catalytically compromised (Q147L) mutation.