Fig. 3
From: Gene editing with ‘pencil’ rather than ‘scissors’ in human pluripotent stem cells
Molecular modules of BEs and PE (A) Base editors consist of nickase Cas9 (nCas9) and deaminase. CBE adopts rAPOBEC deaminase for cytosine deamination. For further improvement, uracil DNA glycosylase inhibitor (UGI) is conjugated. ABE adopt two deaminases (TadA-TadA*) composed of wild type TadA and engineered TadA (TadA*). (B) Editing efficiency and product purity of BEs are continuously improved by optimization of BEs. The original version of CBE (BE3) is further optimized to BE4, BE4max or AncBE4max with additional UGI, codon optimization and/or adoption of ancestor rAPOBEC1 homolog. The original version of ABE, ABE7.10, is optimized to ABEmax by codon optimization and adoption of bis-bpNLS. Further engineering TadA* by PACE or induction of specific mutations (e.g., V106W and D108Q) produces ABE8e and ABE8eWQ. (C) PE is composed of engineered reverse transcriptase (i.e., M-MLV RT) linked to nCas9 and PE guide RNA (pegRNA). M-MLV RT synthesizes DNA strand containing desired edit sequences. The edit strand is inserted into the target sequence. (D) The original version of PE (i.e., PE1) is optimized to PE2 by induction of mutation on M-MLV RT. PE3 is developed by nicking non-editing strand. Co-expression of dnMLH1 with PE2 and PE3, to further improve the efficiency produces PE4 and PE5 respectively. Created with BioRender.com