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

Zhiquan Liu, Siyu Chen, Huanhuan Shan, Yingqi Jia, Mao Chen, Yuning Song, Liangxue Lai, Zhanjun Li

BMC biology 2020 Aug 31

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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.

Citation

Zhiquan Liu, Siyu Chen, Huanhuan Shan, Yingqi Jia, Mao Chen, Yuning Song, Liangxue Lai, Zhanjun Li. Precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions. BMC biology. 2020 Aug 31;18(1):111