## 4 Future Scope of BEs in Other Microbes  
Thus far, efficient BE working systems have been established in lots of microbes. In some, base pairs could be converted to any of the others using a combination of CBE, ABE, and GBE. The microbes that have not been fully established by BEs are listed in Figure 4 and will hopefully be explored in the future. We also believe that the nonmodel microbesbelonging to the same genera of the universal phylogenetic tree in Figure 2, or that are available for CRISPR/Cas9, hold great hope for the future establishment of BEs. The reason why there has been no huge applications of BEs in other microbes is probably due to the novelty of Bes, since they have just been developed in recent years. The toxicity of fusion protein is another reason, but it can probably be solved by replacing nCas9 with dCas9 and decreasing its expression level as mentioned above. The applications of BEs may also be affected by the polyploidy in some species, as multiplex gene editing is not efficient in some microbes; however, BEs still show powerful potential for precise gene editing and genome-wide engineering in other microbes. The primary reasons are as follows. Figure 4: Current development of BEs in different microbial species. (**A**) In theory, any base pair could be converted to any of the others, which only takes three steps at most using the combination of ABE, CBE, and GBE. Firstly, C:G and T:A can be converted to each other by CBE and ABE. Secondly, C:G can be converted to G:C or A:T by GBE. (**a**) If C:G is predominantly converted to A:T by GBE, the most complicated editing step, shaded in grey, is from A:T to C:G, by firstly converting A:T to G:C by ABE, then to T:A by GBE, and finally to C:G by ABE. (**b**) If C:G is predominantly converted to G:C by GBE, the most complicated conversion, shaded in grey, is from A:T to T:A, firstly converting A:T to G:C by ABE, then to C:G by GBE, and finally converting to T:A by CBE. (**B**) The establishment of BEs in different microbes are listed in the table. The achieved base conversions are drawn with a solid line, and base conversions that have not been completed in some microbes are drawn with a dotted line. Firstly, BEs are more applicable than CRISPR/Cas9 for microbes, especially nonmodel microbes. For those species available for CRISPR/Cas9, BEs are very convenient for plasmid construction and transformation. For those unavailable for CRISPR/Cas9, BEs can still achieve editing with high efficiency since the working process does not depend on DSBs and HR [94; 105; 106]. The CRISPR/Cas9 and BEs can also complement gene editing. For example, they are designed as a double-check system to increase the editing efficiency of BEs to 99% at some sites [38]. The inactivation of NHEJ genes by BEs facilitate the further application of CRISPR/Cas9 [69]. The insertions of heterologous metabolic pathways into a genome require CRISPR/Cas9 and in situ mutagenesis to increase the industrial production need for BEs [115]. Moreover, the availability of a BE can cover almost the whole genome in microbes. For example, only 24 genes accounting for 0.77% of the genome in _C. glutamicum_[40], and 32 genes in _B. subtilis_[55], are inaccessible for Cas9 variant-BEs by genome-wide analysis. The mutable and knockout targets for CBE account for 99.8% and 96.6%, respectively, in the genome of _R. sphaeroides_[48]. The editable targets account for 99.83% in _Clostridium ljungdahlii_[72], and 96% of editable stop sites are located in the first 80% of the coding region in _S. melidiot_[75]. For _S. aureus_, almost 68.8% of the genes in MRSA252 strain, and 70.36% of the genes in the Newman strain, possess editable stop sites by CBE [79], and 75% ORF can be inactivated in _M. tuberculosis_[79]. Thirdly, BEs can introduce multiplex random mutagenesis in up to five targets in _Streptomyces_[45] and _B. subtilis_[57] for metabolic pathway engineering. The engineering of _B.