## 6 Promoter Replacement Strategy Using CRISPR-Cas9 Technique  
In 1987, [PERSON] et al. sequenced the characteristic clustered regularly interspaced short palindromic repeats (CRISPR) from E. coli for the first time, but their function was not fully understood [64]. Twenty years later, there was no report about these repeats until in 2007, [PERSON] et al. investigated the function of CRISPR. They found that after a viral infection, bacteria integrate spacers from the phage genome to create CRISPR, combined with Cas protein, that can resist phages by spacer-phage sequence similarity [65]. The fundamental information on the biological role of CRISPR-Cas9 and detailed studies about its mechanism of action and structural aspects have been already explored [66; 67; 68; 69; 70]. Due to its biotechnological application for precise genome engineering it was successfully applied in the mammalian system in 2013 [71]. After that, there was a revolution in the use of the CRISPR system for editing the multiplex genomes in a variety of hosts across all domains of life. The CRISPR-Cas9 system was also used for the multiplex genome editing of _Streptomyces_ strains. The vectors pCRISPomyces-1 and pCRISPomyces-2 derived from suicide vector pKC1139 were widely used in the genome editing of _Streptomyces_. The pCRISPomyces vectors allow the easy assembly of gRNA and editing of templates by Golden Gate assembly or the conventional digestion/ligation method. By using the CRISPR-Cas9 system, the _redN_ from the BGC of undecylprodigiosin, and _actVA-ORF5_ from the BGC of actinorhodin in S. _lividans 66_, _plplD_ and _plpM_ from the BGC of phosphinothricin tripeptide in _S. viridochromogenes_ DSM 40736 were successfully deleted [72]. In addition, they also successfully deleted the full hanthopeptide BGC in S. _albus_ J1074 [72]. Based on the same platform, a pKCcas9 dO vector was constructed for editing the genome of S. _coelicocor_ M145, including the single gene deletion,  
Figure 2: Overview of transcription factor decoy (TFD) strategy. The red arrows represent the promoter. The green arrows represent the reporter gene. as _actII-orf4_, _red_, and _glnR_, and the full BGC of actinorhodin (23.3 kb), undecylprodigiosin (31.6 kb), and Ca\({}^{2+}\)-dependent antibiotic (82.8 kb). The multiplex gene deletion of _actII-orf4_ from the actinorhodin BGC and _redD_ from the undecylprodigiosin BGC was manifested by the co-expression of Cas9 and multi-sgRNA expression modules. Furthermore, the point mutation of _rpsL_ was successfully induced by CRISPR-Cas9 [73]. Therefore, CRISPR-Cas9 represents a time-saving and highly efficient tool for genome editing in _Streptomyces_ strains. The promoter replacement strategy is a very efficient approach for activating silent BGCs in _Streptomyces_. The constitutive promoter as _ermE\({}^{*}\)_ was knocked in on the upstream region of core genes of NRPS gene cluster and PKS-NRPS gene cluster in _S. albus_ J1074, resulting in activation of the blue indigoidine (Figure 3B) and polycyclic tetramate macrolactam family as 6-epi-alteramides A and B (Figure 3B) [74]. Later, the promoter replacement strategy was developed by applying CRISPR-Cas9 to activate the silent gene clusters in streptomyces. KasOp\({}^{*}\) promoter was used for the knock-in experiments to native hosts (Figure 3A) [75; 76]. Ten silent biosynthesis gene clusters in five _Streptomyces_ strains, namely _S. albus_, _S. lividans_, _Streptomyces roseosporus_ NRRL15998, _Streptomyces__venezuelae_ ATCC10712, and _S. viridochromogenes_ DSM 40736, were activated by replacing native promoter by KasOp\({}^{*}\) promoter of the core genes of silent gene clusters. These biosynthesis gene clusters were involved in types I, II, and III PKSs, non-ribosomal peptide synthetase (NRPS), hybrid PKS-NRPS, and phosphonate clusters. The editing efficiency per total screened strains was very high from 50% to 100%, and the efficiency of activation was 100%, whereas a novel pentangular type II polyketide (Figure 3B) was discovered in _S.