Directed Evolution of an Adenine Base Editor withIncreased Activity and Context Compatibility [sgRNA-target library]

Adenine base editors (ABEs) are precise gene-editing agents that convert A:T pairs into G:C through a deoxyinosine intermediate. ABEs function most effectively when the target A is in a TA context. Deficient ABE processing of RA (R = A or G) is most evident when the target A is outside the comfortable editing window or when delivery is suboptimal. In the current study, we report directed evolution of TadA8r, a new variant of the Escherichia coli tRNA-specific adenosine deaminase (TadA) with ultra-fast deoxyadenosine deamination and no context bias. To systematically evaluate the editing performance of ABE8r, we prepared a library wherein target sites were individually paired with their cognate sgRNAs. Similar paired sgRNA-target libraries have previously been used to characterize CRISPR nucleases and base editors. To bridge our high-throughput characterization with future applications of ABEs in disease-relevant contexts, we randomly chose 12,000 NGG-adjacent target sites from the ClinVar database, each hosting a potentially pathogenic G:C-to-A:T transition at protospacer positions 3-10. These G:C-to-A:T transitions are evenly distributed across protospacer positions and XA contexts. The library was stably integrated into HEK293T cells with ABEs transiently expressed to facilitate editing. The entire sgRNA-target cassettes were subsequently amplified and deep sequenced. Consistent editing levels were observed across two biological replicates for all ABEs