Directed Evolution of Improved Zinc Finger Methyltransferases
(A) A schematic of the designed methyltransferase is shown assembled over the new, targeted CpG site. New cognate zinc finger recognition sequences flank a CpG site nested within an FspI site. Zinc fingers CD54-31Opt and CD54a have replaced the HS1 and HS2 zinc fingers. (B) The non-target site contains the HS1 and HS2 zinc finger recognition sites flanking a CpG site nested within a FspI restriction site (i.e. this was the target site in experiments in Figure 2). (C) The relative locations of the target site and non-target site are shown on a plasmid linearized by NcoI digestion. (D) The restriction endonuclease protection assay for methylation at the target and non-target site for the ‘wildtype’ heterodimeric enzyme (KFNSE) and two selected variants with mutations in the region 297–301.