Team:WHU-China/templates/standardpage modelingCas9
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<img src="https://static.igem.org/mediawiki/2013/5/55/WHUFig3cas9.png" /></div> | <img src="https://static.igem.org/mediawiki/2013/5/55/WHUFig3cas9.png" /></div> | ||
<center><em></em>Figure 3. Model prediction compared with data from Fig.5CB of [3]</br></center></br> | <center><em></em>Figure 3. Model prediction compared with data from Fig.5CB of [3]</br></center></br> | ||
- | These data are collected from 1’ end truncation or consecutive mutation experiment of gRNA. In Both figure, as the column number grows, the end truncation/end mutations become more serious, and the total energy of DNA-gRNA binding drops. The prediction of the model is near-linear, but the data show great non-lineality. Obvious platforms formed in the 4-8 column of Fig.2 and column 3-9 of Fig.3, which suggest the gRNA-Cas9 complex is not sensitive for the energy loss cause by the continuous mismatch / truncation at these stage.</br> | + | These data are collected from 1’ end truncation or consecutive mutation experiment of gRNA. In Both figure, as the column number grows, the end truncation/end mutations become more serious, and the total energy of DNA-gRNA binding drops. The prediction of the model is near-linear, but the data show great non-lineality. Obvious platforms formed in the 4-8 column of Fig.2 and column 3-9 of Fig.3, which suggest the gRNA-Cas9 complex is not sensitive for the energy loss cause by the continuous mismatch / truncation at these stage.</br></br> |
On the las part of the model. Kinetic analysis reveals that both concentration and reaction time are important for off-target control.</br></br></br> | On the las part of the model. Kinetic analysis reveals that both concentration and reaction time are important for off-target control.</br></br></br> | ||
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<img src="https://static.igem.org/mediawiki/2013/5/59/WHUDeltaGiCas9.png" /></br></div></br> | <img src="https://static.igem.org/mediawiki/2013/5/59/WHUDeltaGiCas9.png" /></br></div></br> | ||
where N is the total number of phosphates in the duplex, and [Na+] is the total concentration of monovalent cations from all sources (the same equation works for</br> | where N is the total number of phosphates in the duplex, and [Na+] is the total concentration of monovalent cations from all sources (the same equation works for</br> | ||
- | sodium, potassium, and ammonium )over a range of monovalent concentration of 0.05 to1M. </br> | + | sodium, potassium, and ammonium )over a range of monovalent concentration of 0.05 to1M.</br> </br> |
<b>Step6.</b> Calculate △G’ We assume △G’ takes up a form of <img src="https://static.igem.org/mediawiki/2013/d/dc/WHUVectorW.png"/> . Where “a” is an 1×19 vector that contain △G(1) to △G(19) as its value, ω is the weight vector. Only the impact of DNA-gRNA interaction (“a”) is counting as a variable, and the △G contributed by other interaction(eg. protein-DNA interaction) are considered as a constant b. This is also why this model cannot predict Cas9 off-target rate of a target without PAM(NGG), which interact with Cas9 rather than gRNA. (Assumption 4) </br></br> | <b>Step6.</b> Calculate △G’ We assume △G’ takes up a form of <img src="https://static.igem.org/mediawiki/2013/d/dc/WHUVectorW.png"/> . Where “a” is an 1×19 vector that contain △G(1) to △G(19) as its value, ω is the weight vector. Only the impact of DNA-gRNA interaction (“a”) is counting as a variable, and the △G contributed by other interaction(eg. protein-DNA interaction) are considered as a constant b. This is also why this model cannot predict Cas9 off-target rate of a target without PAM(NGG), which interact with Cas9 rather than gRNA. (Assumption 4) </br></br> | ||
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<div style="text-align:center"> | <div style="text-align:center"> | ||
<img src="https://static.igem.org/mediawiki/2013/6/6e/WHUDpdt.png" /></br></div> | <img src="https://static.igem.org/mediawiki/2013/6/6e/WHUDpdt.png" /></br></div> | ||
- | In a typical endonuclease environment, and are always hold. Even in Pattanayak’s paper[1], though the total DNA concentration is 200nM, the concentration every single kind of DNA(with certain sequence) is lower than 0.1nM, which is much lower than KM of any typical restriction enzyme.</br> | + | In a typical endonuclease environment, and are always hold. Even in Pattanayak’s paper[1], though the total DNA concentration is 200nM, the concentration every single kind of DNA(with certain sequence) is lower than 0.1nM, which is much lower than KM of any typical restriction enzyme.</br></br> |
But still, the MM equation remains valid. Because, first, under these conditions, [E] (free E concentration) doesn't change much, because most "enzymes" are in free form and they don't do anything; second, some time after enzyme and substrate are mixed the concentrations of free enzyme sites and of substrate complexed will reach a steady state.[17] </br> | But still, the MM equation remains valid. Because, first, under these conditions, [E] (free E concentration) doesn't change much, because most "enzymes" are in free form and they don't do anything; second, some time after enzyme and substrate are mixed the concentrations of free enzyme sites and of substrate complexed will reach a steady state.[17] </br> | ||
Revision as of 16:05, 27 September 2013
Cas9 Off-target Prediction Model.(Abbreviation: Cas9Off Model)
For a pdf version of the tandem promoter modeling part,click here