Dynamics of single-base editing: Theoretical analysis

Abstract

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.

FIG. 1.

A schematic view of the functioning of the cytosine single-base editor. The gray area indicates the activity window of this BE.

FIG. 2.

Chemical-kinetic model to describe single-base transformations by cytosine BE. The details of the process are discussed in the text. The system starts in state 0, and there are four possible outcomes. State 1 corresponds to no editing events, state 12 describes the editing at both target and bystander sites, state 13 corresponds to the target-site editing (the desired outcome), and state 14 describes the bystander editing. The nucleotides labeled “C” or “T” correspond to cytosine or thymine, respectively, while the nucleotide labeled “X” corresponds to uridine or thymine.

FIG. 3.

Dynamics of different base-editing processes as a function of the binding rate of the deaminase domain to the nucleotide u₁. The following parameters have been used in calculations: u₀ = 1 s⁻¹, u₃ = 1.1 s⁻¹, u₄ = 2.1 s⁻¹, w₀ = 2.9 $*$ 10⁻⁵ s⁻¹, w₁ = 12.54 s⁻¹, and w₂ = 5059 s⁻¹. (a) Mean times for 0 → 5 and 0 → 6 processes in the first stage of base editing. The corresponding probabilities are P_0→5 = 0.321 635 and P_0→6 = 0.000 867. (b) Mean times for 5 → 12, 5 → 13, 6 → 12, and 6 → 14 processes in the second stage of base editing. The corresponding probabilities are P_5→12 = 0.882 299, P_5→13 = 0.117 701, P_6→12 = 0.999 641, and P_6→14 = 0.000 359. (c) Mean times for the overall target editing 0 → 13, bystander editing 0 → 14, and double editing 0 → 12. The corresponding probabilities are P_0→13 = 0.037 856 8, P_0→14 = 3.116 61 * 10⁻⁷, and P_0→12 = 0.284 645.

FIG. 4.

Ratio of probabilities, or fractions of different outcomes of base editing as a function of free-energy perturbation due to mutations. The following parameters have been used in calculations: u₀ = 1 s⁻¹, u₃ = 1.1 s⁻¹, u₄ = 2.1 s⁻¹, w₀ = 2.9 $*$ 10⁻⁵ s⁻¹, w₁ = 12.54 s⁻¹, and w₂ = 5059 s⁻¹.

FIG. 5.

Ratios of mean editing times as a function of free-energy perturbation due to mutations. The following parameters have been used in calculations: u₀ = 1 s⁻¹, u₃ = 1.1 s⁻¹, u₄ = 2.1 s⁻¹, w₀ = 2.9 $*$ 10⁻⁵ s⁻¹, w₁ = 12.54 s⁻¹, and w₂ = 5059 s⁻¹. (a) For unbinding rate, u₁ = 10⁴ s⁻¹. (b) For unbinding rate, u₁ = 10⁵ s⁻¹. (c) For unbinding rate, u₁ = 10⁶ s⁻¹.