ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems

Jingke Xie,Shixue Gou,Yanhui Liang,Zhen Ouyang,Yinghua Ye,Quanjun Zhang,Hui Shi,Liangxue Lai,Longquan Quan,Fangbing Chen,Xiaoping Huang ,Nan Li,Weikai Ge,Kepin Wang,Jiaowei Wang,Meng Lian,Han Wu,Xiaozhu Zhao,Jin Qiu ,Xia Wang,Zanyong Zhuang

doi:10.1186/s12915-020-00866-5

Abstract

BackgroundMany favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Current cytosine or adenine base editors can only accomplish C-to-T (G-to-A) or A-to-G (T-to-C) substitutions in the windows of target genomic sites of organisms; therefore, there is a need to develop base editors that can simultaneously achieve C-to-T and A-to-G substitutions at the targeting site.ResultsIn this study, a novel fusion adenine and cytosine base editor (ACBE) was generated by fusing a heterodimer of TadA (ecTadAWT/*) and an activation-induced cytidine deaminase (AID) to the N- and C-terminals of Cas9 nickase (nCas9), respectively. ACBE could simultaneously induce C-to-T and A-to-G base editing at the same target site, which were verified in HEK293-EGFP reporter cell line and 45 endogenous gene loci of HEK293 cells. Moreover, the ACBE could accomplish simultaneous point mutations of C-to-T and A-to-G in primary somatic cells (mouse embryonic fibroblasts and porcine fetal fibroblasts) in an applicable efficiency. Furthermore, the spacer length of sgRNA and the length of linker could influence the dual base editing activity, which provided a direction to optimize the ACBE system.ConclusionThe newly developed ACBE would expand base editor toolkits and should promote the generation of animals and the gene therapy of genetic diseases with heterogeneous point mutations.

Highlights

Many favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus
Evaluation of the function of ACBE in a HEK293-enhanced green fluorescent protein (EGFP) cell line The PmCDA1 and UGI of Target-activation-induced cytidine deaminase (AID) were fused to the C terminus of adenine base editor (ABE)-7.10, an optimized version of the ABE system, with a nuclear localization signal (NLS) located at the N and C terminals to engineer a new base editor with the abilities of Target-AID and ABE-7.10 (Fig. 1a)
To verify whether the size of base editor expression cassettes (CBE, ABE, and ACBE) influence the expression levels of base editors, base editor expressing vectors fused to the base editor with enhanced green fluorescent protein (EGFP) by P2A were constructed (Additional file 1: Fig. S1a)

Summary

Introduction

Many favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Many desirable agricultural traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms (SNPs) or multiple point mutations with heterogeneous base substitutions at the same locus [2,3,4,5,6,7]. The efficiency of PE for making transition point mutations was reported much lower than that of both CBE and ABE, which makes PE difficult to be used to generate organisms or correct genetic diseases that need base editing of a genome in multiple sites with heterogeneous base substitutions [13,14,15]

Methods

Results

Discussion

Conclusion