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Recent experimental advances led to the development of DNA base editors (BEs) with single-nucleotide precision, which is critical for future progress in various scientific and technological fields. The molecular mechanisms of single-base discrimination, however, remain poorly understood. Using a recently developed stochastic approach, we theoretically investigated the dynamics of single-base editing. More specifically, transient and mean times to edit “TC” motifs by cytosine BEs are explicitly evaluated for correct (target) and incorrect (bystander) locations on DNA. In addition, the effect of mutations on the dynamics of the single-base edition is also analyzed. It is found that for most ranges of parameters, it is possible to temporarily separate target and bystander products of base editing, supporting the idea of dynamic selectivity as a method of improving the precision of single-base editing. We conclude that to improve the efficiency of single-base editing, selecting the probability or selecting the time requires different strategies. Physical–chemical arguments to explain the observed dynamic properties are presented. The theoretical analysis clarifies some important aspects of the molecular mechanisms of selective base editing.
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A general theoretical framework to design base editors with reduced bystander effects
Abstract Base editors (BEs) hold great potential for medical applications of gene therapy. However, high precision base editing requires BEs that can discriminate between the target base and multiple bystander bases within a narrow active window (4 – 10 nucleotides). Here, 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 point mutations at T218 position of A3G-BEs to further reduce its bystander editing. We verify 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.
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- PAR ID:
- 10305229
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 12
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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