Bimodal Adenine and Cytosine CRISPR Base Editing on DNA

Adenine and cytosine base editors (ABEs and CBEs) represent a new genome editing technology that allows the programmable installation of A-to-G or C-to-T alterations on DNA. We engineered Streptococcus pyogenes Cas9-based adenine and cytosine base editor (SpACE) that enables efficient simultaneous introduction of A-to-G and C-to-T substitutions in the same base editing window on DNA. HEK293T cells were transfected with a variety of base editor constructs. nCas9 and GFP expressing cells were negative controls. ABEmax and miniABEmax-V82G served as a control for A-to-G base editing on DNA and A-to-I editing on RNA. Target-AID served as a control for C-to-T base editing on DNA and C-to-U editing on RNA. All base editor constructs co-translationally expressed GFP (P2A-EGFP).The Cells were sorted for GFP based on FITC signal. RNA-seq was performed to measure transcriptional changes associated with different constructs.

腺嘌呤碱基编辑器（adenine base editor, ABE）与胞嘧啶碱基编辑器（cytosine base editor, CBE，合称ABEs与CBEs）是一类新型基因组编辑技术，可对DNA序列进行A→G或C→T的可编程定点修饰。本研究构建了基于化脓性链球菌Cas9（Streptococcus pyogenes Cas9, SpCas9）的双功能腺嘌呤-胞嘧啶碱基编辑器（SpACE），可在DNA的同一碱基编辑窗口内高效实现A→G与C→T碱基的同时定点替换。将多种碱基编辑器表达构建体转染至HEK293T细胞中。表达nCas9与绿色荧光蛋白（green fluorescent protein, GFP）的细胞作为阴性对照。ABEmax与miniABEmax-V82G分别作为DNA层面A→G碱基编辑、RNA层面A→I（腺嘌呤→次黄嘌呤）编辑的阳性对照。Target-AID则作为DNA层面C→T碱基编辑、RNA层面C→U（胞嘧啶→尿嘧啶）编辑的阳性对照。所有碱基编辑器表达构建体均通过P2A自剪切肽共翻译表达增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP，即P2A-EGFP）。基于异硫氰酸荧光素（fluorescein isothiocyanate, FITC）信号对表达GFP的细胞进行分选。通过RNA测序（RNA-seq）检测不同表达构建体对应的细胞转录组变化。