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. Overall design: 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.