## 2 Advancement of BEs in Microbes
### Recent Applications of BEs in Nonmodel Microbes
#### 2.2.1. Industrially Important Nonmodel Microbes  
As a nonmodel yeast with high growth rate, thermotolerance and a wide sugar assimilation spectrum, _Kluyveromyces marxianus_ (_K. marxianus_) has great potential for industrial applications to produce various enzymes, chemicals, ethanol, and so on [81]. The high activity of the NHEJ system in _K. marxianus_ hinders the development of CRISPR/Cas9; therefore, an alternative and efficient gene editing method is required to accelerate its genetic and metabolic characterizations. After Target-AID was established in _S. cerevisiae_[33], it was further applied in _K. marxianus_, belonging to the same class with _S. cerevisiae_, to inactivate NHEJ-related genes, _Nej1_ and _Dnl4_[69]. The NHEJ null mutants with enhanced HR events facilitated the markerless integration mediated by CRISPR/Cas9. Although the mutants were successfully obtained, the editing efficiency of CBE was quite low, which can be improved in the future. With the combination of BEs and CRISPR/Cas9, this nonmodel species can be further explored for industrial applications. _Psedomonas_ is a widespread genus from all over the world, especially found in extreme environments. It is reported to have strong potential for detoxifying environmental pollutants [82] and producing industrial bioactive compounds and pharmaceutical proteins. _Psedomonas putida_ (_P. putida_) was used to produce monoterpene folic acid, fatty alcohols, rhamnoids (Rs), and polyhydroxyalkanoatees (PHAs) [83]. _P. aeruginosa_ and _P. fluorescens_ were reported to be the best-characterized phenazine producers as well [83]. After CBE, rAPOBEC1-nCas9, was proven to be able to inactivate genes in the _Pseudomonas_ species [43], and researchers were committed to improving its editing activity. [PERSON] and colleagues [70] established a developed CBE, fusing rAPOBEC1 and UGI with an enhanced specificity rCas9 variant (eSpCCas9 ppD10A, containing mutations K848A/K1003A/R1060A) to fulfill almost 100% editing efficiency with no detectable gRNA-dependent off-target event in _P. putida_ KT2440, _P. aeruginosa_ PAO1, _P. fluorescens_ Pf-5, and _P. entomophila L48_. Furthermore, similar to previous studies [35; 40], using Cas9 variants to extend editable sites in the genome, eSpCas9 ppD10A was engineered with additional mutations (L1111R, D1135V, G1218R, E1219F, A1322R, R1335A, T1337R), named eSpCas9 ppD10A-NG, recognizing the NG PAM site in _P. putida_ with 100% editing efficiency. rAPOBEC1 was also replaced with YE1[68] to narrow the editing window to nearly 2 nt in case unwanted conversions were created at other cytidine sites and led to low editing efficiency at the target [70]. Variants of deaminase enzymes can also be applied to other microbes to achieve precise conversion when the editing window comprises multiple cytidines. _Streptomyces_, as a well-known antibiotic producer, plays an important role in producing more than two-thirds of medically and agriculturally important secondary metabolites, such as polykeptides and nonribosomal peptides [84]. However, the production of many microbial drugs is very low in original strains since most BGCs are not or poorly expressed under traditional laboratory conditions. The solutions can be knocking out the competitive pathways, replacing negative promoters with strong ones, overexpressing positive regulatory genes [85], etc., which are all manipulated by efficient gene engineering techniques. Primary works have made great developments with CBE and ABE in _Streptomyces_ to achieve single gene inactivation, multiplex gene editing, and reduction of off-target effects by fusing the high fidelity Cas9 variant [44; 45; 46]. Recently, [PERSON] and colleagues [71] developed an antisense RNA (asRNA) interference-enhanced CBE to increase editing efficiency in _Streptomyces lividans 66_ (_S. lividans 66_) since rAPOBEC1-d/nCas9-UGI in _S. lividans 66_ demonstrated a much lower editing efficiency than that in _Streptomyces colicolor_, shown in Table 2. They verified that the deletion of _ung_1 in _S.