Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells

• Published in: Nature biotechnology 2024-10, Vol.42 (10), p.1538-1547
• Main Authors: Ye, Lijun, Zhao, Dongdong, Li, Ju, Wang, Yiran, Li, Bo, Yang, Yuanzhao, Hou, Xueting, Wang, Huibin, Wei, Zhandong, Liu, Xiaoqi, Li, Yaqiu, Li, Siwei, Liu, Yajing, Zhang, Xueli, Bi, Changhao
• Format: Article
• Language: English
• Subjects: 631/61/17/1511; 631/61/201/2110; Agriculture; Bioinformatics; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems - genetics; Cytosine; Cytosine - metabolism; Deoxyribonucleic acid; Directed evolution; DNA; DNA glycosylase; DNA Glycosylases - genetics; DNA Glycosylases - metabolism; E coli; Editing; Escherichia coli - genetics; Gene Editing - methods; Genetic disorders; HEK293 Cells; Humans; Life Sciences; Mammalian cells; Mammals; Mutagenesis; RNA editing; Thymine; Thymine - metabolism; Thymine DNA Glycosylase - genetics; Thymine DNA Glycosylase - metabolism
• ISSN: 1087-0156; 1546-1696; 1546-1696
• DOI: 10.1038/s41587-023-02050-w

Base editors show promise for treating human genetic diseases, but most current systems use deaminases, which cause off-target effects and are limited in editing type. In this study, we constructed deaminase-free base editors for cytosine (DAF-CBE) and thymine (DAF-TBE), which contain only a cytosine-DNA or a thymine-DNA glycosylase (CDG/TDG) variant, respectively, tethered to a Cas9 nickase. Multiple rounds of mutagenesis by directed evolution in Escherichia coli generated two variants with enhanced base-converting activity—CDG-nCas9 and TDG-nCas9—with efficiencies of up to 58.7% for C-to-A and 54.3% for T-to-A. DAF-BEs achieve C-to-G/T-to-G editing in mammalian cells with minimal Cas9-dependent and Cas9-independent off-target effects as well as minimal RNA off-target effects. Additional engineering resulted in DAF-CBE2/DAF-TBE2, which exhibit altered editing windows from the 5′ end to the middle of the protospacer and increased C-to-G/T-to-G editing efficiency of 3.5-fold and 1.2-fold, respectively. Compared to prime editing or CGBEs, DAF-BEs expand conversion types of base editors with similar efficiencies, smaller sizes and lower off-target effects. Base editors lacking a deaminase improve thymine and cytosine conversion.