Figure 1.

The process of gene editing using CRISPR/Cas9 and a BE. (A) Directed by gRNA, CRISPR/Cas9 recognizes the PAM site (5′-NGG-3′, highlighted in red) and introduces DSBs at the target. DSBs induces two cellular DNA repair systems, NHEJ and HR. Through NHEJ, a few imprecise indels are created through error-prone DNA repair. Through HR, the target is replaced by homologous donor DNA with desired edits and is therefore introduced with deletions, insertions, or substitutions of nucleotides. (B) The BE is also directed by gRNA to the target. At the target, deaminase enzyme achieves base conversion without causing DSBs and requiring donor DNA. CBE mediates the conversion of a C:G base pair to a T:A, which can replace four codons (CAA, CAG, CGA, TGG) with premature stop codons (TAA, TAG, TGA) to inactivate the gene. ABE mediates the base conversion from an A:T base pair to G:C, which can replace start codon ATG with GTG or ACG to disrupt the initiation of gene translation. Other than point mutation, BEs can achieve multiplex gene editing, in vivo evolution of protein and strain, metabolic engineering, etc., in various microbes, as discussed in detail below.