A General Theoretical Framework to Design Base Editors with Reduced Bystander Effects

To assist in the design of these optimized editors, we propose a discrete-state stochastic approach to build an analytical model that explicitly evaluates the probabilities of editing the target base and bystanders. Combined with all-atom molecular dynamic simulations, our model reproduces the experimental data of A3A-BE3 and its variants for targeting the TC motif and bystander editing. Analyzing this approach, we propose several general principles that can guide the design of BEs with a reduced bystander effect. These principles are then applied to design a series of new point mutations at T218 position of A3G-BEs to further reduce its bystander editing. We verified experimentally that the new mutations provide different levels of stringency on reducing the bystander editing at different genomic loci, which is consistent with our theoretical model. Thus, our study provides a computational-aided platform to assist in the scientifically-based design of BEs with reduced bystander effects.