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 but it was not in agreement with our software’s predicted experimental outcome.
TALE-M.SssI actively methylates DNA
Figure 2: A TALE-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 control has no T7 polymerase and no possibility of leaky expression. The linearized control is the same band length as blanket methylation.
Our TALE-M.SssI was actively 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 target 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. However, it was unexpected to see methylation skewed in favor of the off target site so we carried out more characterization experiments.
COBRA confirms on and off target methylation
Figure 3: COBRA on induced TALE-M.SssI. The plasmid was bisulfite treated and the target and off target sites were amplified with our standard bisulfite sequencing primers. The amplicons were digested with TaqαI, which only cuts methylated sites, and BamHI, which only cuts untreated DNA.
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. TaqαI digested both the on and off target sites, confirming that the TALE was partially methylating both sites (Figure 3). This validated our assay further, as it reports the same biological outcome as the published COBRA method, but at a fraction of the cost, time, and technical difficulty.
Varied Induction Conditions Clarify TALE Mechanism of Action
Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified. The targeting score (formula below) increases with site-specificity and decreases with enzymatic over activity as measured by the TALE without the binding site.
P = TALE Binding Site
M = No TALE Binding Site
N = On Target Methylation Band
F = Off Target Methylation Band
The software package calculated for us that the largest band we were seeing on the TALE gel was the result of simultaneous target site and off target site methylation while the second largest band was only off target site methylation. We used this information to formulate the Targeting Score to reflect increased site-specificity. We varied induction conditions, expecting one might be more optimal than our previous inductions. As desired, the negative control produced a baseline Targeting Score of almost exactly 1 (1.06). However, no induction condition increased Targeting Score, rather there was a steady decline (Figure 4). This indicated the TALE could be giving negative feedback to the site-specific methylation.
Bisulfite Sequencing Confirms TALE Binding
Figure 5: MaGellin standard bisulfite sequencing primers were used to bisulfite sequence 500 bp including the target site. Rows are individual clones, circles are CpG sites, and distances reflect their distance in bp. Filled in circles are methylated CpGs.
The next step for characterizing functional constructs with MaGellin is to use the standard bisulfite sequencing primers (LINK) for a higher resolution look at individual CpG methylation. We bisulfite sequenced the target site of the induced TALE-M.SssI with the TALE binding site present 4 nucleotides upstream of the target AvaI cut site. The data further validated the enzymatic activity of our TALE-M.SssI in the region of the TALE binding site. It was also the first demonstration that the MaGellin assay and standard primers can easily be used for bisulfite sequencing with a TOPO cloning kit with ~7 day turnaround. Interestingly, no clones were methylated at the CpG site that is within the AvaI site itself (Figure 5).
This data, in combination with the varied induction conditions and COBRA results, led us to hypothesize a new model for the TALE’s mechanism that successfully explains each result. Although 4 nucleotides between the zinc finger binding site and the target cut site was optimal for a published zinc finger fusion with a short linker. That distance is too short for use with a TALE fusion with our linker length. The TALE is at least three times the size of the zinc finger and so TALE binding occludes that CpG from interacting with the methylase.
We have now shown that the novel TALE-M.SssI binds to its binding site strongly, as it almost fully protected that site from the methylase for 24 hours (Figure 4). We have shown that it exhibits methylating activity (Figure 2). Perhaps most interestingly, we have demonstrated that the TALE is large enough to physically occlude neighboring nucleotides from access to its linked effector, which has significant consequences for the recently published slew of TALE fusions – including the TALE-histone methylases, TALE-histone demethylases, and TALE-DNA demethylases for epigenetic engineering. We expect the same result will hold for Cas9-effector fusions, and are in the process of validating that hypothesis. We have already constructed the first dCas9-methylase fusion and demonstrated its enzymatic methylase activity in vivo (Figure 6).
Novel dCas9-M.SssI Methylation Activity Reported by MaGellin
Figure 6: dCas9-M.SssI fusion expressed along with an sgRNA to bind at the target site on MaGellin.
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