From Double-Strand Breaks to Precision Edits: A Comparative Review of Modern Genome Editing Tools

Recent advances in base editing technology and its applications in disease therapy.

Base editors (BEs) are transformative genome engineering tools that enable precise nucleotide substitutions without inducing double-strand breaks (DSBs) or requiring donor DNA templates. Since the first cytosine base editor (CBE) was developed in 2016, the field has advanced rapidly, with the creation of diverse BE variants that incorporate distinct deaminases and glycosylases. These engineered editors have significantly expanded the scope of genome editing by generating deaminated bases or apurinic/apyrimidinic (AP) site lesions, thereby harnessing endogenous DNA repair or replication mechanisms to produce base transitions and transversions. Among these endogenous pathways, trans-lesion synthesis (TLS) plays a particularly critical role in converting AP sites into specific base substitutions. TLS polymerases insert nucleotides opposite AP lesions, and the final editing outcome is dictated by the unique nucleotide preferences of individual TLS polymerases. This review focuses on the action mode of different base editors, highlights their interplays with the TLS, summarizes their potential therapeutic applications and discusses perspective strategies to improve precision and expand targeting scope.

Strategies and mechanisms of precision genome engineering: From gene editing to genome writing

Genomic manipulation has advanced from stochastic nuclease‐mediated disruption toward programmable, deterministic precision. Early clustered regularly interspaced short palindromic repeats (CRISPR) strategies enabled targeted mutagenesis through double‐strand breaks; however, their therapeutic application is limited by genotoxicity, chromosomal instability, and dependence on endogenous repair pathways that are difficult to predict. In this review, we examined the transition from gene editing to genome writing, an approach that decouples genomic modification from host repair pathways to better balance efficiency, precision, and payload delivery. We also discussed the principles of precision technologies, including base and prime editors, and described emerging large‐scale writers, such as CRISPR‐associated transposases and recombinase‐based bridge RNAs, which enable the integration of multi‐kilobase synthetic modules. Beyond enzymatic mechanisms, we further considered the combined use of generative artificial intelligence, structural biology, and novel delivery architectures as potential strategies to overcome current biological limitations. Taken together, these developments point toward Generative Biology, in which computational design and high‐throughput screening transform the genome from a static substrate into a more dynamic model for complex, synthetic functional design.

Efficient and precise programmable DNA knock-in without double-strand breaks.

Design of base editing systems with tunable editing hotspots within a defined window through sgRNA engineering.

Base editors (BEs) enable efficient A-to-G or C-to-T conversions without double-stranded DNA cleavage, but their editing windows remain difficult to tune, limiting genome engineering flexibility. Here, we engineered CRISPR/Cas12b sgRNA by introducing MS2 hairpins to recruit an MS2-N55K-cytidine deaminase-UGI complex, enabling programmable control of the editing window. Three modified sgRNAs were generated by replacing two loop regions, each producing distinct editing hotspots in E. coli. The AID*Δ-MSBE system (sgRNA1.1) generated a window near the PAM with peak activity at C7-C9, while the CDA-MSBE system (sgRNA1.2) produced a distal window with peak activity at C20-C23. Both systems exhibited identical editing patterns in Bacillus subtilis. A dual-orthogonal system (MS2 and PP7) was constructed to simultaneously recruit two deaminase complexes, restoring the classic dCas12b CBE editing pattern. Rifampicin resistance assays confirmed high targeting specificity with low off-target effects. As proof of concept, the MSBEs were successfully employed for the flexible reprogramming of sfGFP fluorescence and the targeted evolution of the endogenous gene rpsE, respectively. Collectively, we developed the MSBEs with tunable editing hotspots, providing innovative tools to enhance the flexibility and accessibility of BEs for genome engineering.

Compact 9dBEs Enable Efficient and Precise Genome Editing in Mammalian Cells and In Vivo

An efficient Cas9d system (Cas9dUltra) is developed through gRNA and protein engineering, and its base editors (9dBEs) further developed through gRNA and protein engineering, enabling efficient and precise genome editing in human cells.

Development and Characterization of RNA Aptamer-Mediated Modular Base Editors Containing Staphylococcus aureus Cas9 Derivatives and Novel Deaminase Orthologs

Base editing enables precise genome modifications without introducing DNA double-strand breaks. Using Streptococcus pyogenes Cas9 as a prototype, we previously developed a modular base editing platform in which the deaminase is recruited by an RNA aptamer engineered into the gRNA, thereby separating sequence recognition from base modification. Here, we expanded this modular base editor toolbox by engineering Staphylococcus aureus Cas9 (SaCas9) in combination with various vertebrate effectors derived from activation induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzyme, catalytic subunit 1 (APOBEC1) orthologs, from bat, lizard, human, and rat. Moreover, we adopted the SaCas9 variants with different protospacer adjacent motif requirements. These base editors generally showed high editing efficiency with low on-target indel formation and low-to-undetectable off-target activities. Quantitative and qualitative differences in editing occur among the base editors when applied to diverse loci, allowing sequence-specific optimization. Together, our study demonstrates the effectiveness of the SaCas9 modular base editors, the robustness of the platform’s modularity, and its feasibility for convenient screening of target-specific base editors.