Directed Evolution of Improved Zinc Finger Methyltransferases
(A) The vector used in selections. The vector encodes for both heterodimeric fragments fused to zinc fingers under the control of separate inducible arabinose (pBAD) and IPTG (lac) promoters, a target site, and the araC gene. (B) A schema of the zinc finger-fused, bifurcated M.SssI and the mutagenized codons used in library construction. Codons corresponding to residues 297–301 of M.SssI (located in the C-terminal fragment) were randomized. Numbering scheme is that of the wildtype M.SssI. (C) An assembled zinc finger-fused heterodimeric M.SssI methyltransferase assembled at the target site. The target site consists of an internal CpG site nested within an FspI restriction site, and flanked by HS1 and HS2 recognition sequences. (D) The non-target site used to assess off-target methylation. The non-target site lacks HS1 and HS2 zinc finger binding sites, but contains a CpG site nested within an SnaBI restriction site (E) An overview of the selections used in this experiment. The schematic illustrates the fates of plasmids encoding inactive methyltransferase (digested by FspI, left), a desired targeting methyltransferase methylated at the target site (not digested, middle) and a nonspecific methyltransferase methylating multiple M.SssI (i.e CpG) sites (digested by McrBC, right).