Construction And Functional Verification Of CRISPRi And CRISPR/Cas9 System In Brucella Melitensis

Brucellosis is an important zoonotic disease that has caused serious economic losses to livestock around the world.It's also a great threat to human health and social safety because of its zoonotic characteristics.However,people didn't have developed a safe and effective vaccine successfully yet.Brucella is the pathogen of brucellosis,to develop a safe and effective vaccine,the research of Brucella gene function is very important.Currently,there are many difficulties in researching Brucella genes.First,Brucella grow slowly,they need about 36-48 hours to be visible on the plate in the stable environment.The wild strains which are separated initial grow more slowly.Second,the brucellosis is a zoonotic disease,making it quite cumbersome and time-consuming to operate the Brucella.These factors all make it difficult to research the gene of Brucella.At present,deleting the gene of Brucella is mainly based on suicide plasmid.However,the method is not only time-consuming and greatly increasing the operational risk of researchers,but also slow the progress of the research.Therefore,it is important to establish a rapid,accurate and effective gene research method in Brucella.CRISPRi and CRISPR/Cas9 technology are developed quickly these days.Because of their accurate and efficient gene editing effect,immediately becoming the focus of many scientists and have been widely used in a variety of biological research.In our study,we investigated the feasibility of applicating CRISPRi and CRISPR/Cas9 system in Brucella.First,we constructed a CRISPRi working plasmid which could be replicated in Brucella.The CRISPRi system consists of two components,a dCas9 protein that inhibits transcription of the target gene and sgRNA that can recognize the target gene.We chose the Brucella outer membrane protein omp19 as the target gene and down-regulated the expression of omp19.Then we evaluated the down-regulation efficiency of omp19 in Brucella.In addition,we also constructed a CRISPR/Cas9 working plasmid that could be replicated in Brucella.After that we used this expression system to try to knock out the omp19 gene in Brucella.The main results are as follows: 1.The expression of omp19 was significant down-regulated by CRISPRi system.Two CRISPRi working plasmids which can be used in Brucella were constructed.One expressed dCas9 protein through constitutive expression pattern and the other through inducible expression pattern.Designing sgRNA which targeting omp19 gene.Then the CRISPRi plasmid were transformed into Brucella 16 M competent cells.The knock down efficiency was analyzed by Real-time Quantitative PCR and western blot.The results showed that the expression of omp19 was down-regulated and significant.2.The Brucella genome was successfully cleaved using the CRISPR / Cas9 system.Constructing the CRISPR/Cas9 working plasmid expressing wtCas9 protein through inducible expression pattern in Brucella.Designing sgRNA which targeting omp19 gene.The plasmid was transformed into Brucella 16 M competent cells.The results showed that CRISPR / Cas9 system has successfully created DSBs in Brucella genome.3.Enhancedthe repair efficiency of non-homologous end joining(NHEJ)pathway in Brucella.Constructing NHEJ working plasmids which expressing Mku and LigD protein in Brucella.Then confirming that the plasmid have enhanced the repair activity of the bacteria.