Team:Penn/MethylaseCharacterization
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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> | 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> | + | </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> | <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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- | <h4><center>TALE-M.SssI | + | <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> | <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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- | 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. | + | 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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<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> | <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> | ||
- | <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. 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). | + | <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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Latest revision as of 03:23, 29 October 2013
Methylase Characterization
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).ZF-M.SssI fully methylates MaGellin plasmid
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). 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
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. Validated COBRA is in agreement with our new MaGellin Assay
Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity
Summary
MaGellin agreed with previously published work that zinc finger methylases are prone to off target methylation.