Establishment And Application Effect Test Of CRISPR System For Prokaryotes Gene Editing

Targeted genome editing,the ability to manipulate a genome precisely,site-specifically and permanently,has been an ideal way to prokaryotic cell genetic improvement.As the third generation of artificial site-specific nuclease,theRNA-guided CRISPR-Cas9 system is becoming a hot spot of the plant biology research,with much simpler design principles,facile construction process as well as the capability of multiplex gene editing.Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated proteins(Cas)systems,found in bacteria and archaea,carry out the adaptive immune responses against invading genetic elements(virus or plasmid)by usingRNAs to guide site-specific cleavage of genetic material.Type II of these systems,using anRNA-guided nuclease(Cas9)to destroy invading DNA,was demonstrated that it can efficiently cleave of any given DNA sequence in tube.Here we configured the well-studied type ? CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/ CRISPR-associated proteins)for targeted genome modification in prokaryotic cell,the main results were as follows:1.we present an engineered CRISPR/Cas system of a suitable for Streptomyces strains.According to Streptomyces rimosus 1139 genome structure and sequence characteristics,we rational design the corresponding components of sgRNA and optimize the core components of the original CRISPR/Cas9 system.Using electroporation way take place conversion of the S.rimosus 1139.The PCR amplification sequencing inspection certificate that using CRISPR/Cas9 system make S.rimosus 1139 gene editing,obtain three different mutations ways:(1)Through verification showed that the transformation of CRISPR/Cas9 system capable of high efficiency in rapid multiplex genome editing of S.rimosus introducing site-directed mutagenesis.The oxytetracycline yield of the mutant strain decreased than the original(2)In order to further improve the efficiency of CRISPR/Cas9 system gene editing,the introduction of creative double sgRNA in the CRISPR components.By optimizing the distance between two sgRNA targeting,introduced in the two sgRNA T7 has terminated and gapdhp(EL)promoter makes two sgRNA complete connection,transformation of electric shock crack S.rimosus after verified by sequencing amplification.Verification results show that the modified CRISPR/Cas9 system can on two targets at the same time the implementation of gene editing,further improve the efficiency of the system of gene editing.The oxytetracycline yield of the mutant strain increased by 23.75% than the original.(3)On the basic of double-basis point mutations,we combined with mutation sequence features introducing homology arms on both sides of the mutation site in the CRISPR/Cas9 systems,electroporation S.rimosus select positive clones amplified target fragment.Verification results show that the system can efficiently on S.rimosus genomic deletions embodiment sentinel.The oxytetracycline yield of the mutant strain increased by 24.83% than the original.2.The establishment of a suitable for gram-negative bacteria CRISPR/Cas9 system.For extending the application scope of CRISPR/Cas9 system,this study through the reasonable design sgRNA components,and its expression in E.coli DH5? mrcA genes to achieve the double locus mutation,established the CRISPR/Cas9 system implemented in gram-negative bacteria gene editing technology,for the use of CRISPR/Cas9 system efficiency,more targeted,high precision edit the E.coli genome provides scientific experimental instruction and data.3.The establishment of a suitable for gram positive bacteria CRISPR/Cas9 system.With Bacillus subtilis ATCC 6633 as experimental strain,construction of plasmid pCas9: : PBSX fixed-point edit PBSX gene and detect its application effect.Test results show that the system can be efficient in B.subtilis genome streamline implementation,so as to explore a new way of streamline of the genome,for using the CRISPR/Cas9 system constructing minimal genome B.subtilis cells to provide reference and experimental basis.