Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination

• Published in: Nature communications 2020-11, Vol.11 (1), p.5827-5827, Article 5827
• Main Authors: Li, Shuo, Yuan, Bo, Cao, Jixin, Chen, Jingqi, Chen, Jinlong, Qiu, Jiayi, Zhao, Xing-Ming, Wang, Xiaolin, Qiu, Zilong, Cheng, Tian-Lin
• Format: Article
• Language: English
• Subjects: 13/31; 42; 42/109; 45/22; 49/90; 49/91; 631/1647/1511; 631/1647/1513/1967/3196; 631/61/201/2110; Adenine; Binding Sites; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; Cytosine - metabolism; Deoxyribonuclease I - genetics; Deoxyribonuclease I - metabolism; Domains; Editing; Gene Editing; HEK293 Cells; Humanities and Social Sciences; Humans; Insertion; Loss of Function Mutation; multidisciplinary; Protein Domains; Protein Engineering - methods; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Ribonucleic acid; RNA; RNA - metabolism; RNA editing; Science; Science (multidisciplinary); Streptococcus infections; Target recognition; Toolkits
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-19730-9

Base editing tools with diversified editing scopes and minimized RNA off-target activities are required for broad applications. Nevertheless, current Streptococcus pyogenes Cas9 (SpCas9)-based adenine base editors (ABEs) with minimized RNA off-target activities display constrained editing scopes with efficient editing activities at positions 4-8. Here, functional ABE variants with diversified editing scopes and reduced RNA off-target activities are identified using domain insertion profiling inside SpCas9 and with different combinations of TadA variants. Engineered ABE variants in this study display narrowed, expanded or shifted editing scopes with efficient editing activities across protospacer positions 2-16. And when combined with deaminase engineering, the RNA off-target activities of engineered ABE variants are further minimized. Thus, domain insertion profiling provides a framework to improve and expand ABE toolkits, and its combination with other strategies for ABE engineering deserves comprehensive explorations in the future. Current SpCas9 adenine base editors that minimise RNA off-target activities have constrained editing windows. Here the authors use domain insertion of TadA into Cas9 to narrow, expand or shift the editing window with RNA off-target minimization simultaneously