## 2 Advancement of BEs in Microbes
### Constructs and Mechanisms of Developed BEs
#### 2.1.3 Latest Development of GBE in Microbes
PegRNA comprises two essential parts: PBS (primer binding site) and RTT (reverse transcriptase template). PE nicks the edited strand. The strand then hybridizes with PBS and extends with the copy of RTT by RT so that the mutation is introduced to the strand. The 5′ flap intermediate is removed by flap endonuclease, and the 3′ flap is ligated. The desired edits are consequently introduced into the DNA sequence. (**D**) GBE consists of nCas9, PmCDA1, and UNG in _E. coli_. GBE deaminates the C at the edited strand and nicks the other strand. U is removed by UNG, so the target becomes an AP site where TLS polymerases assemble and extend the nicked strand by Pol\(\zeta\). When passing by the locus opposite to the AP site, Rev1 cooperates with C and Pol\(\zeta\) with T, which results in the creation of G:C and A:T, respectively, after DNA repair or replication. The different editing preference of GBE in mammalian cells and _E. coli_ attracted [PERSON] and colleagues' attention. They further revealed this mechanism in _S. cerevisiae_[63], as illustrated in Figure 3D. The essential steps for GBE editing are the AP site formation at the edited strand and the nick at the other strand. Then, the replicative DNA polymerases are recruited to repair the nick, known as translesion synthesis (TLS), which are conserved from bacteria to mammals [64]. By knocking out genes encoding TLS polymerases Pol\(\zeta\), Rev1, Pol\(\eta\), and Pol\(\delta\), respectively, Pol\(\zeta\) was proven to work as an extender, Rev1 was shown to specifically insert C at the opposite locus of the AP site, and Pol\(\delta\) was shown to mainly incorporate T or A, whereas Pol\(\eta\) was not involved in this process [63]. The different TLS polymerases working on the AP site led to various base conversions, possibly providing new methods to fuse together polymerases with BEs to accomplish any desired conversions in the future. Recently several base excision repair (BER) proteins including DNA polymerase \(\beta\), DNA ligase III, and XRCC1, were fused separately with rAPOBEC1-nCas9 to manipulate the BER pathway downstream of AP creation, giving rise to G as the major product [65]. [PERSON] and colleagues investigated the impact of DNA repair proteins on GBE efficiency by using CRISPR interference (CRISPRi) screens. They engineered new GBEs with diverse editing profiles by fusing various DNA repair proteins, deaminases, and Cas proteins together [66]. Trained in a library of results at 10,638 genomically integrated target sites, the machine learning models, CGBE-Hive, could predict GBE editing efficiency and purity, as well as bystanders' editing patters with high accuracy. To overcome the limitations of sequence preference and PAM requirement, [PERSON] and colleagues tested a series of new GBEs and argued that the rational design of deaminase, rather than the addition of BER proteins, improved the editing efficiency [67]. The optimized eAID variant-nCas9 fusion protein improved the target compatibility of the GBE system in the GC context [67]. The spCas9 was further replaced with its variants, SpG Cas9 and SpRY Cas9, to expand the targeting range.