Fig 4. Effect of linker length on methylation in the target region as a function of gap length.

Frequency of methylation for cis and trans strand are presented as a function of distance from the PAM (center) and the angle between the dCas9 C-terminus and the MC N-terminus (right) in the structural model for dCas9-MC/MN for a (a) 4-amino acid linker, (b) 15-amino acid linker, or (c) 25-amino acid linker. The angle is defined as that formed between the C-terminus of Cas9 and N-terminus of MC when looking down the DNA axis from the CpG site towards the PAM (i.e. the angle formed in the plane of the page in the view of Fig 2b and 2d). Full amino acid sequences of linkers can be found in Fig 1a. The dotted line indicates the fraction of the target sites that are methylated on at least one strand assuming that cis and trans strand methylation is independent. For c, methylation frequency data was not obtained for the trans strand for the gap lengths of 31 and 37 bp. Data in (b) is from Xiong et al. [13].