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.
Abstract
Adenine base editors (ABEs), which enable A•T-to-G•C base editing, have emerged as a powerful tool with potential therapeutic applications. However, conventional ABEs suffer from bystander nucleotide conversions, limiting their utility for precise editing. Here we present a single-nucleotide resolution ABE (snuABE) created by fusing a nickase Cas9, nCas9-H840A, with the deaminase domain of ADAR (adenosine deaminase acting on RNA), which acts on DNA:RNA hybrids, instead of TadA, which acts on single-stranded DNA in conventional ABEs. snuABE requires a target-adenine guide RNA (tagRNA) that introduces a mismatch at the target adenine, enabling highly specific A-to-G editing by ADAR. Engineering ADAR from Pediculus humanus using the in silico protein evolution algorithm EvolvePro, along with 3'-end protection of the tagRNA, enhanced snuABE activity, yielding a median efficiency of 5.4% and a maximum efficiency of 50.0% across 32 targets in HEK293T cells. snuABE exhibits no detectable DNA off-target editing at predicted off-target or orthogonal R-loop sites, highlighting its potential as a precise and safe base-editing technology.
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.
Adenine base editors (ABEs) are CRISPR effectors that introduce A-T to G-C transitions in the genome using a nucleotide deaminase fused to a Cas protein. ABEs have been evolved to have very high editing efficiency, but off-target editing effects compromise their precision and hinder their applications. Here we explore the activity and specificity relationship of ABEs using a combination of machine learning-guided design and high-throughput screening. We designed a diverse library of 12,000 variants and built quantitative bacterial selection systems that allowed us to simultaneously measure their on-target and off-target editing. We 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, or genotoxicity. These results were supported by in vitro studies and consistent with editing experiments in mammalian cells. Finally, the activity and specificity trade-offs were recapitulated among previously reported engineered variants and a selection of library variants spanning the activity spectrum. Our results suggest that fundamental architectural improvements will be necessary to transcend the activity and specificity limitations for the next generation of ABEs.
Maria Lukarska, L. Oltrogge, Hunter M. Nisonoff et al.· bioRxiv· 1 citation
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.
Current dual base editors and hypermutators exhibit limited types of base conversion and constrained mutational diversity. To address this challenge, we engineer a compact dual base editor “A&GBE” by fusing a deaminase and glycosylase with nickase Cas9 (nCas9) to enable concurrent adenine and cytosine editing. Furthermore, we develop quadruple base mutating modules (QBMM) by fusing nickase or dead Cas9 with engineered thymine-DNA glycosylase (TDG), N-methylpurine DNA glycosylase (MPG) and TadA8e, together with MS2-MCP system recruited activation-induced cytidine deaminase (AID). This QBMM enables simultaneous mutation of all four types of nucleotide bases (A, T, G, C) within an approximately 100 base pair (bp) sequence surrounding the gRNA target site. This platform generates hyper diverse multi-nucleotide variants, establishing a powerful tool for accelerated protein evolution, functional genomics, and disease modeling.
Na Zhao, J. S. Olajide, Zhihong Guan et al.· Nature Communications· 0 citations
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 are demonstrated.
J. Collantes, Kellen Xu, M. Ruiz-Urigüen et al.· The CRISPR Journal· 0 citations
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, Tongyun Luo et al.· Biotechnology Advances· 0 citations