Methylase Characterization

For a detailed, graphical explanation of the MaGellin work flow, please download the MaGellin Workflow Specifications Sheet, which includes all of the steps in the MaGellin workflow.

We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.

The process further validated our MaGellin assay:

1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.
2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.
3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.

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 replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.

TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay

MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.

Validated COBRA is in agreement with our new MaGellin Assay

Given the novel nature of our MaGellin assay, we wanted to see if a traditional, published methylation assay would be in agreement about the TALE-M.SssI result.

We bisulfite converted the plasmid and used our validated bisulfite sequencing primers on both the target and off target site, then used the COBRA assay. The controls recapitulated that our primers are biased for only bisulfite converted DNA, as desired. Unconverted DNA would have been digested by the control enzyme, otherwise. Methylation-sensitive TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites, as MaGellin reported (Figure 3). This validated our assay further, as MaGellin reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.

Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity

Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its in vivo. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis.