Precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions
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RESEARCH ARTICLE
Open Access
Precise base editing with CC contextspecificity using engineered human APOBEC3G-nCas9 fusions Zhiquan Liu1†, Siyu Chen1†, Huanhuan Shan1, Yingqi Jia1, Mao Chen1, Yuning Song1, Liangxue Lai1,2,3,4* and Zhanjun Li1*
Abstract Background: 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. Results: 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 eA3GBE 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. Conclusions: These novel base editors with improved precision and CC context-specificity will expand the toolset for precise gene modification in organisms. Keywords: CRISPR/Cas9, Base editor, eA3G, Precision
Background The clustered regularly interspaced short palindromic repeat (CRISPR) system has exhibited powerful genome manipulation capability in various organisms [1]. Base editor, a revolutionary technology derived from the CRIS PR system, is composed of a cytidine deaminase or an evolved adenine deaminase fused to nCas9 and enables the conversion of C·G to T·A or A·T to G·C base pair in organisms, respectively [2, 3]. In contrast to conventional gene-editing nucleases, CBE represents significant * Correspondence: [email protected]; [email protected] † Zhiquan Liu and Siyu Chen contributed equally to this work. 1 Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin University, Changchun 130062, China Full list of author information is available at the end of the article
advances in precise genome manipulation since it can achieve targeted C-to-T conversions without generating DNA double-strand breaks (DSBs) or requiring a donor template, and it induces lower levels of unwanted insertion/deletion mutations (indels) [2, 4]. The most commonly used CBE architecture, rA1-BE, consists of rat APOBEC1 (rA1) fused to a Streptococcus pyogenes Cas9 (SpCas9) nickase [2]. Efficient editing by rA1-BE requires the target C within a ~ 5-nucleotide window near th
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