Precise DNA base editing using AlphaFold3-based contact modelling

Key Points:

• dI-profiling and other off-target detection methods reveal overlapping and unique off-target sites for HEK4 and HBG_sg-198 sgRNAs, with targeted amplicon sequencing validating editing rates and dI-profiling signals at these sites in HEK293T cells.
• AF3-based analysis shows that single-nucleotide mismatches at specific spacer positions induce localized changes in Cas9-DNA-RNA interactions, with high-fidelity Cas9 variants exhibiting reduced protein-nucleic acid contacts at mutated residues compared to wildtype.
• The Cas9(K1020D) mutation significantly reduces genome-wide sgRNA-dependent off-target editing by 69-83% compared to ABE8e, as shown by dI-profiling and molecular dynamics simulations indicating altered residue-DNA interactions without compromising on-target editing efficiency.
• ContactSeek framework, integrating AlphaFold3-derived contact probabilities and RNA-seq off-target data, identifies key residues in TadA8e and APOBEC3A deaminases that modulate RNA off-target editing, enabling design of variants with improved specificity and reduced transcriptome-wide off-target effects.
• Cas12a variants with mutations such as R284E and R284E-H29D demonstrate substantial reductions (up to 95%) in genome-wide gRNA-dependent off-target editing compared to wild-type Cas12a-A3A, while maintaining effective on-target editing in HEK293T cells.