Mathematical modeling is a core part of Waterloo iGEM: we have nearly as many team members typing furiously away in our dry lab as we do wrangling transformations in our wet lab. This year, we created models in Python, MATLAB and NetLogo that examine the feasibility of our design and provide tools for assessing future designs. The code for each model is available on our GitHub and details on the formulation of each model may be found in the pages linked below.
Cas9 Frameshift Dynamics
PAM Structural Bioinformatics
A key limitation of the current use of S. pyogenes Cas9 for gene editing is the inability to target sequences which are not adjacent to a NGG PAM sequence. To overcome this issue, we developed a pipeline to probe Cas9 mutants with altered PAM specificity using simulation tools in PyRosetta and Python. A detailed description of the analysis and software used in this work can be found on the Modeling Engineered Cas9 PAM Flexibility.
The simulation managed to successfully predict the PAM specificity of wild type spCAs9. However, validation against known Cas9 mutants was less successful, predicting a mix of correct and incorrect, though biochemically similar, PAM sequences.