This work systematically map domain insertion sites within the high-activity TadA8e adenine base editor using structure-guided design and computational inference and finds that TadA8e accepts domain insertions at multiple surface sites, with overall activity and editing window width dependent on insert position rather than domain identity.
Abstract
Adenine base editors (ABEs) enable efficient A•T to G•C conversion, but their broad activity windows frequently cause unintended bystander edits. We hypothesized that insertion of a bulky, inert protein domain into the base editor would limit the effective reach of the deaminase, thereby preferentially directing editing to the intended target adenine. Here, we systematically map domain insertion sites within the high-activity TadA8e adenine base editor using structure-guided design and computational inference. We find that TadA8e accepts domain insertions at multiple surface sites, with overall activity and editing window width dependent on insert position rather than domain identity. Excitingly, inserting domains at residue L68 preserved robust editing across multiple genomic loci while tightly focusing the editing window around position 5. Insertion of superfolder GFP at this site produced a base editor variant with a narrower editing window, the ability to track edited cells by fluorescence, and markedly reduced Cas-independent off-target editing. Our work highlights domain insertion engineering as a powerful strategy to create more focused and precise CRISPR base editors.
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.
This work found that the ABEs were fully described by the single dimension of intrinsic deaminase activity with no evidence for independent specialization with respect to local sequence context, editing window width, RNA editing, RNA editing, or genotoxicity.
Maria Lukarska, L. Oltrogge, Hunter M. Nisonoff et al.· bioRxiv· 1 citation
This review compares Cas9-mediated homology-directed repair (HDR) with generations of cytosine base editors (CBE1–CBE3), adenine base editors (ABE1-ABE7), and prime editors (PE1–PE3b), focusing on their mechanistic distinctions, efficiencies, delivery challenges, and therapeutic applications.
Anoushka Sinha· American Journal of Student...· 0 citations
A single-nucleotide resolution ABE created by fusing a nickase Cas9, nCas9-H840A, with the deaminase domain of ADAR, which acts on DNA:RNA hybrids, instead of TadA, which acts on single-stranded DNA in conventional ABEs.
Hyeon Woo Im, Bada Jeong, Yeji Lee et al.· Nature Biotechnology· 1 citation
Abstract Traditional adenine base editors (ABEs), primarily derived from TadA, suffer from bystander editing and limited diversity, while artificial intelligence (AI)-designed deaminases such as Deam-P32 exhibit lower efficiency and precision than state-of-the-art editors such as ABE8e. Here, we report the OpenABE variants through the structure-guided evolution of Deam-P32 by optimizing DNA engagement and the base-contacting pocket and incorporating C-terminal extensions from ABE8e. These variants achieved a 16–36-fold increase in A-to-G conversion efficiency over Deam-P32, matching the editing efficiency of ABE8e across 29 endogenous nuclear loci while mitigating bystander cytosine editing. OpenABEs also reduced ATC motif bystander editing and produced fewer guide RNA-independent off-target effects on DNA and RNA. Further, we adapted these variants to mitochondrial DNA editing by designing OpenABE-TALEDs, which yielded editing efficiencies comparable or superior to those of ABE8e-TALEDs. Delivery through engineered virus-like particles further enhanced specificity and product purity. These results demonstrate that structure-guided refinement of AI-designed deaminases can produce precise, versatile base editors for nuclear and mitochondrial genomes, expanding the research and therapeutic applications of genome editing toolkits.
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.
Rui Tao, Min Li, Tong-Yun Luo et al.· Biotechnology Advances· 0 citations
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