Difference between revisions of "Team:Waterloo/Modeling/Cas9 Dynamics"
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<section id="motivation" title="Motivation"> | <section id="motivation" title="Motivation"> | ||
<p>CRISPR/Cas9 has been extensively studied for its applications in eukaryotic genome editing and gene expression control. Last year, the <a href="https://2014.igem.org/Team:Waterloo/Math_Book/CRISPRi">Waterloo iGEM team</a> created an ODE model of dCas9 binding and control of gene expression. This year, however, the modelling team chose to investigate the effects of CRISPR/Cas9 on an genomic rather than molecular level. Specifically, we wanted to model the <strong>accumulation of mutations</strong> in a target genome and <strong>eventual deactivation</strong> of target genes after cutting by CRISPR/Cas9 and repair by Non-Homologous End Joining (NHEJ).</p> | <p>CRISPR/Cas9 has been extensively studied for its applications in eukaryotic genome editing and gene expression control. Last year, the <a href="https://2014.igem.org/Team:Waterloo/Math_Book/CRISPRi">Waterloo iGEM team</a> created an ODE model of dCas9 binding and control of gene expression. This year, however, the modelling team chose to investigate the effects of CRISPR/Cas9 on an genomic rather than molecular level. Specifically, we wanted to model the <strong>accumulation of mutations</strong> in a target genome and <strong>eventual deactivation</strong> of target genes after cutting by CRISPR/Cas9 and repair by Non-Homologous End Joining (NHEJ).</p> | ||
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| + | <p>The Cas9 nuclease creates double-stranded breaks (DSBs) 3-4 bp upstream of the PAM site adjacent to its sgRNA <cite ref="Addgene_CRISPR2015"></cite>. In the absence of a template, the DSBs will be repaired by Non-Homologous End Joining (NHEJ), which is an error-prone process that sometimes creates indels at the site of repair <cite ref="Betermier2014"></cite>. This effect has recently been exploited to target double-stranded viruses such as HBV <cite ref="Dong2015"></cite><cite ref="Seeger2014"></cite>. Though there have been extensive effort to characterize the factors that contribute to effective targeting and deactivation by CRISPR/Cas9 and NHEJ, they have not, to the best of our knowledge, been incorporated into a single tool. This model aims to synthesize the characterixa</p> | ||
</section> | </section> | ||
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<section id="formation" title="Model Formation"> | <section id="formation" title="Model Formation"> | ||
<h2>Model Formation</h2> | <h2>Model Formation</h2> | ||
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| + | <h3>Probability of Double-Stranded Cuts made by CRISPR/Cas9</h3> | ||
| + | <p>Taking into account target effects</p> | ||
| + | <h3>Possible Outcomes After a Double-Stranded Break</h3> | ||
| + | <h3>Error-Prone Repair by NHEJ</h3> | ||
| + | <p>Indel Probabilities</p> | ||
| − | <h3> | + | <h3>Large Deletions</h3> |
| − | <h3> | + | <h3>Other Model Parameters</h3> |
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<p> Talk about where all the probabilities come from </p> | <p> Talk about where all the probabilities come from </p> | ||
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</div> | </div> | ||
</div> </section> | </div> </section> | ||
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| + | <h3>Framework and Pseudocode</h3> | ||
<section id="results" title="Results"> | <section id="results" title="Results"> | ||
Revision as of 05:11, 17 September 2015
Modelling Genomic Effects of CRISPR/Cas9
CRISPR/Cas9 has been extensively studied for its applications in eukaryotic genome editing and gene expression control. Last year, the Waterloo iGEM team created an ODE model of dCas9 binding and control of gene expression. This year, however, the modelling team chose to investigate the effects of CRISPR/Cas9 on an genomic rather than molecular level. Specifically, we wanted to model the accumulation of mutations in a target genome and eventual deactivation of target genes after cutting by CRISPR/Cas9 and repair by Non-Homologous End Joining (NHEJ).
The Cas9 nuclease creates double-stranded breaks (DSBs) 3-4 bp upstream of the PAM site adjacent to its sgRNA . In the absence of a template, the DSBs will be repaired by Non-Homologous End Joining (NHEJ), which is an error-prone process that sometimes creates indels at the site of repair . This effect has recently been exploited to target double-stranded viruses such as HBV . Though there have been extensive effort to characterize the factors that contribute to effective targeting and deactivation by CRISPR/Cas9 and NHEJ, they have not, to the best of our knowledge, been incorporated into a single tool. This model aims to synthesize the characterixa
Model Formation
Probability of Double-Stranded Cuts made by CRISPR/Cas9
Taking into account target effects
Possible Outcomes After a Double-Stranded Break
Error-Prone Repair by NHEJ
Indel Probabilities
Large Deletions
Other Model Parameters
Talk about where all the probabilities come from
Framework and Pseudocode
Results
Model Validation
Include notes on how the model matches reality/our expectations of reality in this section.
Simulate w/ targets that mismatch to different extents.
Effect of sgRNA Strength
Matt visualizations for different sgRNAs.
Graph of 3 different sgRNA designs of different strengths, show % functional
Importance of Large Deletions
Include notes on how the model matches reality/our expectations of reality in this section.
Effect of Cas9 Concentration
Include notes on how the model matches reality/our expectations of reality in this section.
Predicting CRISPR Plant Defense
This model was applied to the CRISPR Plant Defense aspect of our project, investigating whether the P6 protein of Cauliflower Mosaic Virus (CaMV) could be deactivated by frameshift mutations. The P6 protein was chosen as a focus of the investigation because it suppresses natural plant RNAi defenses and trans-activates translation of other CaMV proteins . Details on P6 and the CaMV genome can be found on CaMV Biology page.
The model was run with HOW MANY targets in the P6 gene of the simulated CaMV genome described above. We tracked the percent of simulated genomes with functional P6 across 1000 runs fo the model, giving a general prediction of how long it will take before the P6 of a particular CaMV genome is rendered non-functional by our Plant Defense system.
PLOT % functional for P6/time over many simulations.