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.
Abstract
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.
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, Melany Ruiz-Urigüen et al.· The CRISPR Journal· 0 citations
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.
A yeast selection platform is developed to engineer Cas9 with re-specified activity across multiple additional non-canonical PAMs in yeast, further demonstrating its utility as a general and programmable framework for expanding the therapeutic reach of precision genome editing.
Julia Tartaglia, Vivian Nguyen, John James Desmarais et al.· bioRxiv· 0 citations
The engineered strain achieved a marked increase in 1,4-butanediamine production, demonstrating that promoter editing via ABE can effectively regulate metabolic flux and enhance the production of the target product, as well as serving as a valuable reference for the biosynthesis of other high-value-added chemicals.
Yan-Ling Sun, Dicheva Ma, Ke Zhao et al.· Microorganisms· 0 citations
Base editors hold great promise in endogenous mutagenesis for genetic screening. However, the development of base editors that induce saturated multi-base conversions with diverse mutation spectrum is challenging. Here, we develop triple base editors (smACGs) that simultaneously mutagenize adenine, cytosine, and guanine within the same allele. Through screening and embedding engineered deaminase and alkyladenine DNA glycosylase variants in Cas9 structure, smACGmax is generated to catalyze robust triple-base conversion efficiencies of up to 41% across varied sequence contexts while maintaining low RNA off-target effects compared to previous dual-base editors. We apply smACGmax to enable high coverage (94%) of targeted HBEGF mutagenesis that identified diphtheria toxin-resistant mutations and to dissect SF3B1 variants with alternative splicing specificity via complex single, double, and triple base conversion screening. smACGmax expands base conversion capability from single and double substrates to trinucleotide level, which facilitates the generation of high-diversity and complex genetic variants, providing a useful platform for mutagenesis-based application. Broad-spectrum base mutagenesis at the same endogenous loci with base editors remains a challenge. Here, the authors developed smACGmax to catalyze efficient multi-base conversions across adenine, cytosine and guanine, and enable high-diversity functional screening in HBEGF and SF3B1 variants.
