Now that the MaGellin assay was validated for non-specific methylases, we were ready to test site-specific methylases. These are fusion proteins, a DNA binding domain linked to a methylase by a serine glycine chain. We used the prokaryotic methylase M.SssI for all of our studies (BBa_K1128000).
We wanted to first recapitulate published results with a zinc finger binding domain, and then characterize our two novel site-specific methylases: using the TALE and CRISPR/Cas binding domains.
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Zinc Finger-M.SssI Fusion
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay does everything we need it to do.
SHOW ZINC FINGER DATA
Figure 1: The ZF-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.
To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).
TALE-M.SssI Fusion
TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are quickly replacing zinc fingers. We performed a similar experiment with our TALE-M.SssI fusion, with and without the binding site present at the target cut site. We ran the gel and saw a significant effect on the methylation pattern. At first, the activity seemed similar to the zinc finger but it was not in agreement with our software’s predicted experimental outcome.
TALE-M.SssI actively methylates DNA
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