| Over the past decades,occurrence of multidrug resistant strains closely associated with the overuse of antibiotics,super pathogen are found worldwide,a variety of new diseases continue to emerge,however,screening of natural products,particularly microbial products has fallen out of favor.Recent advances in genomic sequencing have revealed a wealth of information about potential secondary metabolite pathways.And recent progress in several aspects of natural-product research and microbial genomics suggests that the potential of natural-product diversity and discovery is vastly underestimated.On one hand,in vitro cloning and editing of the biosynthesis gene cluster can avoid the establishment of genetic manipulation system,on the other hand,it is helpful to promote the research of the strain which cannot be cultured under laboratory conditions or the gene cluster which cannot express the target chemical compound.Therefore,this kind of technology plays an important role in fully exploiting the potential of biosynthesis gene cluster.Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein(CRISPR/Cas)is an acquired immune defense system which is widely existed in prokaryotes and can help the host resist the invasion of mobile genetic elements(MGEs).Due to the simplicity and efficiency of type Ⅱ CRISPR/Cas9 system,worldwide researchers gradually focus on this field.And Streptococcus pyogenes Cas9(SpyCas9)is the most widely used Cas9,which is a kind of RNA-guided endonuclease that allows SpyCas9 to specifically target and cleave DNA double-stranded by the pairing of RNA and DNA bases.Because of the target binding and cleavage requires only three elements—SpyCas9,mature crRNA and DNA substrate containing PAM,people have developed it into a kind of efficient genomics research toolbox and has been used successfully in almost all model organisms.In this study,we aims to establish a new and efficient gene cluster cloning and editing tool in vitro by combination of SpyCas9 and appropriate technologies.Firstly,in order to establish a new and efficient gene cloning and editing tool in vitro,we express SpyCas9 protein in the laboratory,and successfully established the CRISPR/SpyCas9 in vitro digestion system,providing technical support for following research.By using the combination of established SpyCas9 in vitro digestion system and Gibson assembly.We proposed SpyCas9-Gibson assembly(CGA)strategy and successfully cloned the Agglomerin A biosynthesis gene cluster directly from the genome of Enterobacter agglomerans PB-6042,the following product analysis shown that CGA strategy works well.For the sake of improving the efficiency of cloning large gene cluster,multi-steps CGA strategy was established.Eventually,an unknown gene cluster X was directly cloned from Shewanella psychrophila WP2 genomic DNA by using multi-steps CGA strategy.Which provides an effective in vitro cloning strategy for large fragment or discontinuous gene cluster cloning.In order to further optimize the multi-steps CGA strategy,we chose to combine the SpyCas9 in vitro digestion system and TAR cloning and established SpyCas9-TAR cloning strategy.Through an appropriate design,the cloning efficiency of TAR should be significantly improved.By using this strategy,Sisomicin biosynthesis gene cluster has been cloned directly from cosmids of Micromonospora inyoensis DSM46123.Moreover,the intermediates Gentamicin A and Sisomicin were also observed by the heterologous expression and detection for the first time.These results not only verify SpyCas9-TAR strategy is a robust approach to clone large or discontinues gene cluster,but also provides an important model for the research of Sisomicin biosynthesis gene cluster.For the study of editing large gene cluster in vitro,we demonstrated that nucleotide trimming is specific to the noncomplementary strand of the blunt end without PAM,in circular dsDNA as well as in linear dsDNA,and that this seriously compromises the accuracy of subsequent cloning.By using a simple end repair procedure,the ICE system greatly increase the likelihood of accurate cloning.Furthermore,we have demonstrated that even large biosynthetic gene clusters(20-38 kb)can be rapidly edited with high efficiency and accuracy by ICE.The ICE system has the potential for highthroughput production of a series of engineered DNAs,thus laying the foundation for systematically knocking out every single gene in biosynthetic gene clusters in vitro. |
