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

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

doi:10.1186/s12915-020-00849-6

Abstract

BackgroundCytidine 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.ResultsWe 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.ConclusionsThese novel base editors with improved precision and CC context-specificity will expand the toolset for precise gene modification in organisms.

Highlights

Cytidine base editors (CBEs), composed of a cytidine deaminase fused to Cas9 nickase, enable efficient C-to-T conversion in various organisms
The significantly reduced base editing efficiencies of engineered human APOBEC3G (eA3G)-BE were observed in 6 target sites with non-CC contexts compared with that of rat APOBEC1 (rA1)-BE (Additional file 1: Fig. S2)
The eA3G-BE showed reduced off-target base editing compared with rA1-BE, consistent with previous report of engineered human APOBEC3A (eA3A)-BE [9] (Additional file 1: Fig. S3). These results suggested that the eA3G-BE can induce site-dependent lower or similar base editing efficiency compared to that of rA1-BE, maintaining distinct preference for CC context in human cells

Summary

Introduction

Cytidine base editors (CBEs), composed of a cytidine deaminase fused to Cas nickase (nCas9), enable efficient C-to-T conversion in various organisms. It negatively affects the precision of targeted base editing, which are not ideal for precise disease modeling and gene therapy where accurate single C substitution is required [6] To overcome this limitation, optimized rA1 with mutant deaminase domains (YE base editors) or shortened linker between rA1 and nCas has been used to narrow the editing window in human cells [7, 8]. An engineered human APOBEC3A (eA3A) domain with TCR (R = A/G) context-specificity has been reported to efficiently reduce bystander mutations, and it has been proven to be superior to conventional base editors with narrowed window in the TCR motifs [9, 10] Contextdependent base editors, such as eA3A-BE, represent an important direction that offers precise base editing, while the application of them was restricted by the presence of TCR motifs [9]. Many precise base editors exist currently, it is still difficult to achieve accurate editing in target sites with multiple Cs

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Conclusion