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Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing
Nature Communications ( IF 14.7 ) Pub Date : 2020-09-25 , DOI: 10.1038/s41467-020-18715-y
Minh Thuan Nguyen Tran 1 , Mohd Khairul Nizam Mohd Khalid 1 , Qi Wang 1 , Jacqueline K R Walker 1 , Grace E Lidgerwood 2, 3 , Kimberley L Dilworth 4 , Leszek Lisowski 4, 5 , Alice Pébay 2, 3 , Alex W Hewitt 1, 6
Affiliation  

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.



中文翻译:

工程域镶嵌 SaCas9 腺嘌呤碱基编辑器,减少了 RNA 脱靶,增加了在靶 DNA 编辑

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,

更新日期:2020-09-25
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