## 2 Advancement of BEs in Microbes
### Constructs and Mechanisms of Developed BEs  
#### 2.1.1 Latest Development of CBE and ABE in Microbes  
The basic constructs and working mechanisms of CBE and ABE are illustrated in Figure 3A,B. CBE in Figure 3A is named Target-AID, fusing Petromyzon marinus CDA1 (PmCDA1) to the C terminus of d/nCas9 (d/nCas9-PmCDA1). The editing window of Target-AID is commonly at the positions \(-16\) to \(-20\) (counting the PAM as positions 1-3), highlighted in dark red in Figure 3. It is important to note that another type of CBE,  
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, GGA, 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. not shown in Figure 3, fuses rat APOBEC1 (rAPOBEC1) to the N terminus of d/nCas9 (rAPOBEC1-d/nCas9) with the editing window at \(-13\) to \(-17\). In past years, various versions of CBE and ABE have been developed to overcome the editing limitations, such as low editing efficiency, off-target effects, and PAM requirements, by utilizing Cas9 homologs and variants, deaminase homologs and variants, modifying gRNA, and so on. Their characteristics and applications in animals, plants, and bacteria are concluded comprehensively by excellent reviews [27; 30; 32; 50; 51; 52]. [PERSON] and colleagues also introduced a decision tree for choosing different BEs on the basis of several criteria [38]. Recently, several developed BEs have been reported to further address editing limitations in microbes. For example, PmCDA1 was engineered with intensive truncations, and several mutations were inlaid in the middle of nCas9 [53], which not only performed comparable editing efficiency to Target-AID, but also greatly minimized the off-target effect and molecular size in _S. cerevisiae_. The gRNA structure was engineered with a bubble hairpin that contained a \(5^{\prime}\) extended sequence that was complementary to the guide sequence in order to decrease the off-targets of both CBE and ABE in _E. coli_[54]. In _Bacillus subtilis (B. subtilis)_, CBE has been developed from a primary version dCas9-PmCDA1 with a narrow editing window (\(-17\) to \(-18\)) [55] compared with dCas9-PmCDA1-UGI [56], with almost 100% editing efficiency at positions \(-16\) to \(-20\) by adding UGI and replacing the strong promoter _Pgrac_ with a weak one, _Pspac_. Furthermore, to overcome the low transformation of nCas9-PmCDA1 [36; 55; 56], which is probably caused by the toxicity of the fusion protein, the PmCDA1-nCas9-UGI was integrated into the genome and performed with approximately 100% efficiency at positions \(-15\) to \(-20\)[57]. The 20 nucleotides (nt) gRNA was also engineered using extensions or adding an artificial stem loop to increase the editing efficiency and to expand the editable window [57]. The development of CBE and ABE to overcome their limitations in microbes are all concluded in Table 1.  
C.G and T.A are currently interchangeable through the combination of CBE and ABE, which is able to generate 62 different amino acid substitutions [44].