Kinetic modeling of the CRISPR-Cas9 genome editing system

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Professor Shi-Jie Chen of the University of Missouri-Columbia is supported by an award from the Chemical Theory, Models and Computational Methods program in the Division of Chemistry. Dr. Chen develops computational and theoretical methods to investigate the physical mechanisms for the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- CRISPR-associated protein 9 (Cas9) genome editing process. CRISPR-Cas9 genome editing is an increasingly powerful tool with widespread applications beyond all our expectations. However, the physical principles governing the CRISPR gene editing process are unclear and as a result, the rational design and quantitative prediction for gene editing remain a significant challenge. Dr. Chen and coworkers will employ physical and chemical theories to uncover the kinetic mechanism of CRISPR gene editing and establish the quantitative relationship between the kinetic properties of the system and the gene editing efficiency. The research result, deployed through freely accessible software and web servers, will lead to new, accurate designs of CRISPR-Cas9 genome editing and impact CRISPR applications from better genetic manipulation of crop species to more precise gene therapy for various diseases. On the educational and outreach front, this research will lead to new materials for curriculum development, create multi-disciplinary training opportunities for students and postdoctoral scholars, and in the long term, train our next generation leaders in theoretical and computational chemistry and biology. At the center of this research is the development of a kinetic model for CRISPR-Cas9 gene editing system. In the first part of the project, to find the critical kinetic step in genome editing, Dr. Chen and coworkers will use a random forest method to extract the key ingredients from the large data sets collected from various experiments. In the second part of the project, by focusing on the critical kinetic steps, they will employ statistical mechanical theory to model kinetic pathways of the R-loop formation, the critical step in gene editing kinetics. In the third part of the project, Dr. Chen and coworkers are testing, refining, and validating the model using experimental data. The result of this research will be a new model capable of predicting the kinetic mechanism of the CRISPR-Cas9 system, such as kinetic pathways, transition states, kinetics intermediates, and rate constants, and for a given CRISPR-Cas9 system, estimating the genome editing efficiency for both on- and off-target editing.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.