Effect of linker length on methylation targeting in ER2267 <i>E</i>. <i>coli</i> cells.

(a) Schematic of protein constructs and linkers used in E. coli studies. (b) Methylation was tested in a two plasmid ER2267 system with inducible dCas9-MC/MN genes and constitutively expressed sgRNA1. Each of site 1 and site 2 contains a CpG site imbedded in an FspI site to allow screening of methylation by FspI endonuclease. Plasmid DNA isolated from cells containing this plasmid pair and expressing sgRNA1 were subjected to in vitro restriction enzyme protection assay to test (c) the effect of shortening the linker length on methylation at a fixed gap distance (d) the effect of lengthening the linker on methylation at longer gap distances, and (e) the effect of lengthening the linker on methylation at gap distances for which the dCas9-MC/MN enzyme with the 15 aa linker is ineffective at methylation. sgRNA1 is designed to target methylation to site 1. The sizes of bands corresponding to methylation at the indicated sites are specified to the right of the gel images. The asterisk indicates that dCas9-MC/MN expression was not induced. All plasmid DNA was digested with SacI (to linearize the plasmid) and FspI (to test for methylation at sites 1 and 2). Plasmid pEncode lacks FspI restriction enzyme site and is the band at the top of the gel.