Precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions

• Published in: BMC biology 2020-08, Vol.18 (1), p.111-111, Article 111
• Main Authors: Liu, Zhiquan, Chen, Siyu, Shan, Huanhuan, Jia, Yingqi, Chen, Mao, Song, Yuning, Lai, Liangxue, Li, Zhanjun
• Format: Article
• Language: English
• Subjects: Animals; APOBEC-3G Deaminase - genetics; APOBEC-3G Deaminase - metabolism; Base editor; Cells (Biology); Context; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR/Cas9; Cytidine deaminase; Cytidine Deaminase - genetics; eA3G; Editing; Editors; Efficiency; Embryos; Gene Editing; Genetic modification; Genetically modified organisms; Genomes; Humans; Mutation; Nonsense mutation; Nucleotides; Precision; Rabbits
• ISSN: 1741-7007; 1741-7007
• DOI: 10.1186/s12915-020-00849-6

Cytidine base editors (CBEs), composed of a cytidine deaminase fused to Cas9 nickase (nCas9), enable efficient C-to-T conversion in various organisms. However, current base editors can induce unwanted bystander C-to-T conversions when multiple Cs are present in the ~ 5-nucleotide activity window of cytidine deaminase, which negatively affects their precision. Here, we develop a new base editor which significantly reduces unwanted bystander activities. We used an engineered human APOBEC3G (eA3G) C-terminal catalytic domain with preferential cytidine-deaminase activity in motifs with a hierarchy CCC>CCC>CC (where the preferentially deaminated C is underlined), to develop an eA3G-BE with distinctive CC context-specificity and reduced generation of bystander mutations. Targeted editing efficiencies of 18.3-58.0% and 54.5-92.2% with excellent CC context-specificity were generated in human cells and rabbit embryos, respectively. In addition, a base editor that can further recognize relaxed NG PAMs is achieved by combining hA3G with an engineered SpCas9-NG variant. The A3G-BEs were used to induce accurate single-base substitutions which led to nonsense mutation with an efficiency of 83-100% and few bystander mutations in Founder (F0) rabbits at Tyr loci. These novel base editors with improved precision and CC context-specificity will expand the toolset for precise gene modification in organisms.