Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain

• Published in: Nature cell biology Vol. 22; no. 6; pp. 740 - 750
• Main Authors: Zhang, Xiaohui, Chen, Liang, Zhu, Biyun, Wang, Liren, Chen, Caiyu, Hong, Mengjia, Huang, Yifan, Li, Huiying, Han, Honghui, Cai, Bailian, Yu, Weishi, Yin, Shuming, Yang, Lei, Yang, Zuozhen, Liu, Meizhen, Zhang, Ying, Mao, Zhiyong, Wu, Yuxuan, Liu, Mingyao, Li, Dali
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.06.2020; Nature Publishing Group
• Subjects: 13/1; 13/31; 14/34; 38/23; 38/44; 45/22; 45/41; 45/77; 631/136/1425; 631/1647/1511; 631/337/1427; 64/60; AC generators; Animals; Binding proteins; Biomedical and Life Sciences; Cancer Research; Cell Biology; Cell Differentiation; Cell lines; Comparative analysis; Conversion; CRISPR-Cas Systems; Cytidine - chemistry; Cytidine - genetics; Deoxyribonucleic acid; Developmental Biology; DNA; DNA-binding protein; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Editing; Editors; Embryo, Mammalian - cytology; Embryo, Mammalian - metabolism; Embryos; Female; Gene Editing; Genetic diversity; Genetic variance; HEK293 Cells; Hemoglobin; Humans; Insertion; Life Sciences; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mutation; Nucleotide sequence; Protein binding; Protein Domains; Proteins; Rad51 protein; Rad51 Recombinase - genetics; Rad51 Recombinase - metabolism; Single-stranded DNA; Single-stranded DNA-binding protein; Stem Cells; technical-report; Thymidine
• ISSN: 1465-7392; 1476-4679; 1476-4679
• DOI: 10.1038/s41556-020-0518-8

Cytidine base editors are powerful genetic tools that catalyse cytidine to thymidine conversion at specific genomic loci, and further improvement of the editing range and efficiency is critical for their broader applications. Through insertion of a non-sequence-specific single-stranded DNA-binding domain from Rad51 protein between Cas9 nickase and the deaminases, serial hyper cytidine base editors were generated with substantially increased activity and an expanded editing window towards the protospacer adjacent motif in both cell lines and mouse embryos. Additionally, hyeA3A-BE4max selectively catalysed cytidine conversion in TC motifs with a broader editing range and much higher activity (up to 257-fold) compared with eA3A-BE4max. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations in the haemoglobin gamma gene promoter to mimic a naturally occurring genetic variant for amelioration of β-haemoglobinopathy, suggesting the therapeutic potential of the improved base editors. Li and colleagues report base editor variants with improved targeting efficiencies and broader editing windows by fusing the original base editors with the single-stranded DNA-binding domain of Rad51.