Team:Penn/MethylaseCharacterization

From 2013.igem.org

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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.</br> </br>
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.</br> </br>
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The process further validated our MaGellin assay:  
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<h7>The process further validated our MaGellin assay:</br>
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</br>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.
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</br>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.</br>
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</br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with bisulfite sequencing that the TALE exhibited targeted inhibition of the methylase. This has serious implications for the multitude of TALE-effector systems that have recently been developed: the TALE can inhibit the effector if the linker length and distance between the TALE binding site and target site are not optimized. The MaGellin workflow is well suited to solve this optimization problem.
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</br>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.</br>
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</br>3. We demonstrated the enzymatic activity of our novel dCas9-M.SssI and are now characterizing it further.
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</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.</h7>
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<h4>Zinc Finger-M.SssI Fusion</h4>
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<h1>Zinc Finger-M.SssI Fusion</h1>
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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.
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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 (Xu et al., 1997).<h7> MaGellin agreed with previously published work</h7>, from various groups with no common, standardized assay. It's possible MaGellin is more sensitive to off target methylation than their assays because <h7>MaGellin only reports site-specific methylation if 100% of the methylations is site-specific for that plasmid.</h7>
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</br><h4><center>ZF-M.SssI fully methylates MaGellin plasmid</center></h4>
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/6/60/Zf-102813.png" alt="Workflow" width="400" ><figcaption><i>Figure 1: A ZF-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 (N) control has no T7 polymerase and no possibility of leaky expression. The linearized control (L)  is the same band length as blanket methylation.</i></figcaption></figure></div>
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<font color="red">SHOW ZINC FINGER DATA</font>
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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. MaGellin reported off target activity, irrespective of zinc finger binding (Figure 1). <h7>MaGellin is sensitive to off target effects</h7> because there is no background CpG methylation in E.coli and the plasmid is short compared to a mammalian genome. We are interested in examining this initial finding further to determine its reproducibility and relevance to previously published zinc finger-methylases.
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</br><i>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.</i></br></br>
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<h1>TALE-M.SssI Fusion</h1>
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</br>TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct (Cong et al., 2012). 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 with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.
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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).
 
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<h4><center>TALE-M.SssI partially methylates on and off target sites on MaGellin plasmid</center></h4>
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/2b/91213-Induced-Tale-2.png" alt="Workflow" width="600" ><figcaption><i>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.</i></figcaption></figure></div>
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<h4>TALE-M.SssI Fusion</h4>
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</br>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 digestion pattern, demonstrating the methylation activity of our novel fusion protein, but it was not in full agreement with our software’s predicted experimental outcome.
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MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. The off target methylation was significantly reduced compared to what MaGellin reported for the zinc finger fusion. <h7>This showed MaGellin can detect changes in the site-specificity of methylation due to swapping DNA binding domains</h7>. We still 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.
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<b><center>TALE-M.SssI actively methylates DNA</center></b>
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<center><h4>Validated COBRA is in agreement with our new MaGellin Assay</center></h4>
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/1/1a/91213_Induced_Tale-2.pdf" alt="Workflow" width="600" ><figcaption><i>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.</i></figcaption></figure></div>
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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. </br>
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="300px"><figcaption><i>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. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.</i></figcaption></figure></div>
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We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. 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). <h7>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 (Xiong et al., 1997).</h7>
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</br>
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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.
 
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<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4>
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<center><b>COBRA confirms on and off target methylation</center></b>
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Given the quick turnaround and cost-effectiveness of the MaGellin assay, <h7>it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its fuction <i>in vivo</i></h7>. 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 (less than 5% the cost of bisulfite sequencing).
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/95/FinalCOBRATaleInd.png/514px-FinalCOBRATaleInd.png" alt="Workflow" width="400px"><figcaption><i>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.</i></figcaption></figure></div>
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>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 and normalized by background intensity. The plot is missing data for samples marked *. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div>
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We varied induction conditions, expecting one might be more optimal than our initial inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase was still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site.
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</br>This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. <h7>Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.</h7>
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<b>Up Next:</b> We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.
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We bisulfite converted the plasmid and used our <a href="https://2013.igem.org/Team:Penn/AssayValidation">validated bisulfite sequencing primers</a> on both the target and off target site, then used the <a href="https://2013.igem.org/Team:Penn/AssayOverview">COBRA assay</a>. 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.
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<center><b>Varied Induction Conditions Clarify TALE Mechanism of Action</center></b>
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/8/8c/3dplot-Converted.png/723px-3dplot-Converted.png" alt="Workflow" width="600px"><figcaption><i>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. </i></figcaption></figure></div>
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P = TALE Binding Site</br>
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M = No TALE Binding Site</br>
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N = On Target Methylation Band</br>
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F = Off Target Methylation Band</br></i>
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/f/fa/CodeCogsEqn42314.gif" alt="Workflow" width="250px"><figcaption><i> </i></figcaption></figure></div>
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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.
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<center><b>Bisulfite Sequencing Confirms TALE Binding
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<h1>Summary</h1>
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</center></b>
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</br>
</br>
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<h7>MaGellin was designed to optimize the development of robust tools for site-specific methylation.</h7> To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.
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<p></br>MaGellin agreed with previously published work that zinc finger methylases are prone to off target methylation. <h7>We were able to construct and test this construct in the three weeks after regionals because of MaGellin's simple workflow.</h7>
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/2/23/TaleIndOn102513-1.png" alt="Workflow" width="500px"><figcaption><i>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. </i></figcaption></figure></div>
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<p></br><h7>We picked up on the noisiness of our TALE-M.SssI using MaGellin,</h7> which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. <h7>Importantly, we could not have reached this result without MaGellin,</h7> because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal.<h7> Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites,</h7> along the lines of how TALE-Nucleases cleave DNA (Li et al, 2007).
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The next step for characterizing functional constructs with MaGellin is to use the <a href="https://2013.igem.org/Team:Penn/AssayValidation">standard bisulfite sequencing primers </a> 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).
 
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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.</br></br>
 
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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).
 
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<h4>Novel dCas9-M.SssI</h4>
 
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</br> <center>dCas9-M.SssI reports methylation activity with our MaGellin assay</center>
 
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/7/75/Cas9_Induction2.png" alt="Workflow" width="200px"><figcaption><i>Figure 6: dCas9-M.SssI fusion expressed along with an sgRNA to bind at the target site on MaGellin. The larger band in the induced sample reports methylase activity.</i></figcaption></figure></div>
 
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<p></br>We started work on the dCas9-M.SssI and have already seen promising activity in the initial screen. We are now pursuing further characterization with our MaGellin assay.
 
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<h4>Summary</h4>
 
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MaGellin was developed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our workflow.
 
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<p>To gain our new insight into a fundamental shortcoming of recently developed genome engineering tools, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, bisulfite sequencing, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA. That could be the best way to construct epigenetic engineering tools with the specificity necessary for clinical applications.
 
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<p>Moreover, we have demonstrated the importance of studying the distance between the binding site and the target site, and shown the ideal distance will be very different between different DNA binding domains. This boils down to an optimization problem between choosing binding sites and linker lengths; this is exactly the sort of problem that the MaGellin system is designed to solve in a fast and affordable manner.
 
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<center><a href="https://2013.igem.org/Team:Penn/MethylaseOverview">&#8592;Previous</a> <a href="https://2013.igem.org/Team:Penn/MaGellinFutureDirections">Next&#8594;</a></center>
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Latest revision as of 03:23, 29 October 2013

Penn iGEM

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 (Xu et al., 1997). MaGellin agreed with previously published work, from various groups with no common, standardized assay. It's possible MaGellin is more sensitive to off target methylation than their assays because MaGellin only reports site-specific methylation if 100% of the methylations is site-specific for that plasmid.

ZF-M.SssI fully methylates MaGellin plasmid


Workflow
Figure 1: A ZF-M.SssI was cloned into and expressed from MaGellin, then induced with IPTG in T7 Express cells. The NEB10 (N) control has no T7 polymerase and no possibility of leaky expression. The linearized control (L) is the same band length as blanket methylation.

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. MaGellin reported off target activity, irrespective of zinc finger binding (Figure 1). MaGellin is sensitive to off target effects because there is no background CpG methylation in E.coli and the plasmid is short compared to a mammalian genome. We are interested in examining this initial finding further to determine its reproducibility and relevance to previously published zinc finger-methylases.

TALE-M.SssI Fusion


TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct (Cong et al., 2012). 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 with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.

TALE-M.SssI partially methylates on and off target sites on MaGellin plasmid


Workflow
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.

MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. The off target methylation was significantly reduced compared to what MaGellin reported for the zinc finger fusion. This showed MaGellin can detect changes in the site-specificity of methylation due to swapping DNA binding domains. We still 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.
Workflow
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. In COBRA, as opposed to MaGellin, digestion means methylation, and no digestion means no methylation.

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 (Xiong et al., 1997).

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 fuction 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 (less than 5% the cost of bisulfite sequencing).
Workflow
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 and normalized by background intensity. The plot is missing data for samples marked *. The dotted white circle marks the conditions of our initial experiments.
We varied induction conditions, expecting one might be more optimal than our initial inductions. As expected, the "without binding site" negative control showed significant off target effects, presumably because the TALE did not bind and the methylase was still active. However, as we induced for longer times and with more IPTG than we had originally used, MaGellin reported off target methylation increasing even for the TALE with the binding site.
This makes sense, as the TALEs could saturate the binding sites on a low copy plasmid or genome. We expect this has implications for recently published TALE-effector systems, which may be noisier than expected as it is much more difficult to assay off-target effects in mammalian systems. We propose the use of split-reconstitution systems that are activated by co-localization of effector subunits at a target site. Our future direction is to design this sort of chimeric TALE, cloning and screening the construct will be quick and easy with MaGellin. Notably, we would not have noticed the off target methylation so quickly on a mammalian genome, but it was simple to de-noise this complex system with MaGellin.

Up Next: We have designed and cloned a dCas9-M.SssI fusion, which we will characterize with MaGellin.

Summary


MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.


MaGellin agreed with previously published work that zinc finger methylases are prone to off target methylation. We were able to construct and test this construct in the three weeks after regionals because of MaGellin's simple workflow.


We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns. Importantly, we could not have reached this result without MaGellin, because the one-plasmid system in a noiseless chassis makes it simple, even unavoidable, to detect off target methylation. Conversely, for the previously published work in mammalian systems, it was not as feasible to detect off target effects across a long genome with background signal. Based on our data, future improvements on genome engineering tools should include the construction of two targeted fusions with subunits of effectors that only dimerize and show activity at the binding sites, along the lines of how TALE-Nucleases cleave DNA (Li et al, 2007).

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