Engineering precise adenine base editors with infinitesimal bystander mutation and off-target editing

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications with the development of highly efficient and compatible adenine deaminase TadA-8e. However, they also induce bystander mutations and Cas9 independent off-target editing. Here we generated a serial of ABE variants through structure-guided rational design of TadA8e. ABE8e with a N108Q mutation, named as ABE9, significantly reduces both adenines and cytosines bystander editing. Introduction of an additional mutation, such as L145T or F84T, further refines the ABE to catalyze A-to-G conversions within 1-2 nucleotides editing window or even specifically at A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites in cells and mouse embryos. ABE9s induce very minimal RNA and undetectable Cas9-independent DNA off-target effects. Due to the minimized editing window, ABE9s could further broaden the targeting scope for precise correction of pathogenic SNPs when fused to PAM-expanded Cas9 variants.