Discovery of deaminase functions by structure-based protein clustering

• Published in: Cell 2023-07, Vol.186 (15), p.3182-3195.e14
• Main Authors: Huang, Jiaying, Lin, Qiupeng, Fei, Hongyuan, He, Zixin, Xu, Hu, Li, Yunjia, Qu, Kunli, Han, Peng, Gao, Qiang, Li, Boshu, Liu, Guanwen, Zhang, Lixiao, Hu, Jiacheng, Zhang, Rui, Zuo, Erwei, Luo, Yonglun, Ran, Yidong, Qiu, Jin-Long, Zhao, Kevin Tianmeng, Gao, Caixia
• Format: Article
• Language: English
• Subjects: base editing; context preference; CRISPR-Cas Systems; Cytidine Deaminase - genetics; Cytidine Deaminase - metabolism; Cytosine - metabolism; Ddd; deaminase; DNA; Gene Editing; protein classification; Proteins - metabolism; Sdd; specificity; structural prediction
• ISSN: 0092-8674; 1097-4172; 1097-4172
• DOI: 10.1016/j.cell.2023.05.041

The elucidation of protein function and its exploitation in bioengineering have greatly advanced the life sciences. Protein mining efforts generally rely on amino acid sequences rather than protein structures. We describe here the use of AlphaFold2 to predict and subsequently cluster an entire protein family based on predicted structure similarities. We selected deaminase proteins to analyze and identified many previously unknown properties. We were surprised to find that most proteins in the DddA-like clade were not double-stranded DNA deaminases. We engineered the smallest single-strand-specific cytidine deaminase, enabling efficient cytosine base editor (CBE) to be packaged into a single adeno-associated virus (AAV). Importantly, we profiled a deaminase from this clade that edits robustly in soybean plants, which previously was inaccessible to CBEs. These discovered deaminases, based on AI-assisted structural predictions, greatly expand the utility of base editors for therapeutic and agricultural applications. [Display omitted] •AI-guided structural classification establishes new deaminase family relationships•SCP1.201 deaminase clade contains both ssDNA and dsDNA cytidine deaminases•Newly deaminases are smaller and have increased activities and minimal off-targets•Further AI-assisted truncation enables AAV packaging and efficient soybean editing AI-assisted structural predictions and alignments establishes a new protein classification and functional mining method, further discovering a suite of single- and double-stranded deaminases, which show great potential as bespoke base editors for therapeutic or agricultural breeding applications.