## 2 Advancement of BEs in Microbes
### Constructs and Mechanisms of Developed BEs
#### 2.1.3 Latest Development of GBE in Microbes  
nCas9-CBE has been reported to obtain quite a few C to non-T products in _S. cerevisiae_[33], _C. glutamicum_[39], _R. shareoides_[48], _Klebsiella pneumoniae (K. pneumoniae)_[60], and so on. Inspired by the observations above, [PERSON]'s group [61] and [PERSON]'s group [62] fused UNG to the C terminus of BE4 max (an improved version of CBE with two UGIs and biparticle SV40 NLS) and rAPOBEC1-nCas9, respectively, to amplify the effect on product impurities. The results both showed a great preference for C-to-G conversion in mammalian cells. [PERSON] and colleagues further optimized the GBE system with the APOBECEC1 variant (R33A) to reach 41.7-71.5% editing efficiency [61]. In the meantime, [PERSON] and colleagues reported that fusing UNG to the N terminus of nCas9-AID (UNG-nCas9-AID), shown in Figure 3D, preferred to convert C to A in _E. coli_ with an average editing specificity of 93.8% \(\pm\) 4.8%, and an editing efficiency of 87.2% \(\pm\) 6.9% [62]. Figure 2: Phylogenetic tree of all microbes that have been researched by BEs, based on small-subunit rRNA sequences and constructed using the maximum-likelihood estimation. The phylogenetic tree includes 21 genera and six classes which are shadowed in different colors. (**A**) Microbes belong to the kingdom Fungi, phylum Ascomycota, and class Saccharomyces. _Saccharomyces_ and _Kluyveromyces_ belong to the same family, Saccharomyces, highlighted in brown. _Yarrowia_ and _Aspergillus_ belong to the same family, Dipodascaceae, highlighted in yellow. (**B**) Microbes belong to the kingdom Bacteria, phylum Proteobacteria, and class Alphaproteobacteriabacteria. _Rhodobacter_ and _Brucella_ belong to the family Rhodobacteraceae and Rhizobacterae, respectively. _Agrobacterium_ and _Sinorhizobium_ belong to the same family, Brucellaceae, highlighted in purple. (**C**) Microbes belong to the kingdom Bacteria, phylum Proteobacteria, and class Gammaproteobacteria. _Pseudomonas, Acinetobacter_, and _Shewanella_ belong to the family Pseudomonasaceae, Moraxellaceae, and Shewanellaceae, respectively. _Escherichia_ and _Klebsiella_ belong to the same family, Enterobacteriaceae, highlighted in pink. (**D**) Microbes belong to the kingdom Bacteria, phylum Actinobacteria, and class Actinomyceta. _Streptomyces_, _Mycobacterium_, and _Corynebacterium_ belong to the family Streptomycetaceae, Coryneb bacteriaceae, and Mycobacteriumae, respectively. (**E**) Genus _Clostridium_ belongs to the kingdom Bacteria, phylum Firmicutes, class Clostridia, and family Clostridiaceae. (**F**) Microbes belong to kingdom Bacteria, phylum Firmicutes, and class Bacilli. _Penaithacillus_, _Penaithacillus_, _Staphylococcus_, and _Bacillus_ belong to the family Paenibacillusae, Lactobacillus, Staphylococcus, and Bacillaceae, respectively. Figure 3: The basic components and working mechanisms of CRISPR-mediated base editing systems. The common editing windows are highlighted in dark red in the DNA sequence. (**A**) CBE consists of cytosine deaminase (PmCDA1 in (A)), impaired Cas9, gRNA, and UGI (inhibits uracil DNA glycosylase, abbreviated to UNG, to improve the efficiency of CBE but is not essential). Under the R-loop, CBE deaminates C to U. If UNG removes U, the apurinic/apyrimidinic (AP site) will soon be reversed to C. If UGI is added to CBE, U will be retained at the locus where DNA polymerase will read U as T. The C-G is successfully conversed to T.A. If dCas9 is replaced with nCas9, the nonedited strand will be nicked. Without the inhibition of UGI, U will be removed at the other strand, where AP endonuclease nicks the edited strand and leads to DSBs, bringing indels or cell death. (**B**) ABE consists of impaired Cas9, gRNA, and ecTadA–TadA’ homodimer (ecTadA: wild-type tRNA adenosine deaminase from _E. coli_, TadA’: evolved ecTadA that can operate on DNA). ABE deaminates A to 1 at the target, where I is misread as G by DNA polymerase. (**C**) PE consists of nCas9, reverse transcriptase (RT), and prime editing gRNA (pegRNA).