Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.

随着包括胞嘧啶碱基编辑器和腺嘌呤碱基编辑器 (ABE) 在内的 CRISPR/Cas 碱基编辑系统的开发，精密基因组工程取得了显着进步。在此，我们比较了循环排列和域嵌入 Cas9 碱基编辑器的编辑概况，发现在它们的域内插入后，在很大程度上保持了靶向编辑，但 ABE 的结构排列会影响不同的 RNA 脱靶事件。凭借这种洞察力，结构引导设计被用于设计 SaCas9 ABE 变体（microABE I744），与当前的 N 端连接的 SaCas9 ABE 变体相比，该变体显着提高了靶向编辑效率并减少了 RNA 脱靶足迹。这代表了可用的最小的 AAV 可交付 Cas9-ABE 之一，