Application Of Gene Editing System In Brucella,etc Pathogenic Microorganisms

Brucella is facultative intracellular,Gram-negative bacteria that infect animals and humans,and cause Brucellosis.Brucellosis is characterized by abortion and infertility in animals,as well as arthritis and undulant fever in human beings.The infection caused by Brucella leads to severe economic losses and public health problems.Macrophages are primary target cells of Brucella.Once invasion,the transport of Brucella is facilitated by lipid raft.Brucella has developed stealth strategies to survive in host cells,and remodel intracellular environment,including involving in regulating apoptosis,autophagy and immune response.Brucella survives and replicates in safe niche that named Brucella-containing vacuoles,and releases from cells at last stage.The establishment of intracellular parasitism makes prevention,controlling and therapy of Brucellosis difficult.The previous studies indicated that micro RNAs were involved in host gene expression regulation in response to pathogen invasion.The expression profiles of mi RNA were impacted as mammalian cells were stimulated by pathogenetic bacteria,such as Samonella enteria,Listeria monocytogenes,and Mycobacterium spp.In present study,mi RNA expression profile of RAW264.7 cells in response to Brucella melitensis infection was explored through high-throughput sequencing.The differentially expressed mi RNAs were achieved through bioinformatic analysis,so as their putative targets.The predicted interaction between mi R-1981 and Bcl-2 was validated by dual-luciferase assay.Bcl-2 protein plays a vital role in regulating both apoptosis and autophagy activities,which induces autophagy and represses apoptosis.In addition,mi RNA-like RNAs originated from Brucella were observed in Brucella-infected library.In order to confirm them,we sequenced the small size RNAs extracted from in vitro cultured Brucella melitensis bacterial cells,analyzed the biogenesis and validated the one mi RNA-like RNA through northern blotting.To better understand mechanism of Brucella spp surviving in macrophage,we established CRISPR-guided base-editing method for genome manipulation.The editor mainly consists of cytidine deaminase-Cas9 fused protein and sg RNA.This method can convert cytosine（C）to uracil（U）at targeted sites without double-strand breaks,resulting in C to T substitution after DNA repair during replication.Mediated by the Base-Editor,the Gln（CAA）et al can be induced into STOP（TAA）et al codons,which provides an alternative approach to knock-out target genes.Our results demonstrated that:（1）the third generation Base-Editor（BE3）edited single-copy genes in Escherichia coli with high efficiencies（nearly 100%）;（2）the efficiencies decreased to approximately 50%while BE3 was used to modify multiple copy（3～5copy/cell）gene possessed in plasmid;（3）the editing efficiencies depended on r APOBEC1-n Cas9-UGI protein expression,which decreased in the situation of low nutrition.Continuously subculture accumulated edited bacterial cells in the whole E.coli population;（5）the Pseudorabbies virus genome in bacterial artificial chromosome（BAC）can also act as substrate for Base-Editor in E.coli;（6）Brucella-encoded vir B10 gene was mutated using BE3 editor.For manipulations in Brucella melitensis genome,we found that“escaper”occurred while r APOBEC1-n Cas9-UGI was expressed at low level,and that the edited bacterial cells could not be accumulated through passaging.This indicated that a suspected anti-CRISPR system may exist in Brucella melitensis.Transcriptome of escapers were explored through high-throughput sequencing.The reported anti-CRISPR proteins were identified from bacteriophage genomes.However,none of putative prophage genes were differentially expressed compared to non-targeting control.Furthermore,the conserved differentially expressed genes among diverse kinds of escapers were several genes related to metabolism,which may suggest that metabolism component may contribute to inhibition on CRISPR.To avoid that inhibition,improve fidelity and broaden PAM recognition,we constructed Hi Fi-BE2-NG editor.Compared to BE3 editor,the Hi Fi-BE2-NG is capable of editing gene with NGN PAM with low frequencies.However,the edited E.coli cells accumulated in the whole population after continuously passaging.Therefore,we came up with an optimal protocol for base-editing using Hi Fi-BE2-NG.Last but not least,an alternative gene editing system was constructed,which was based on Retron operon,λBet and CRISPR/Cas9 system.The editing efficiency could be 10^-3.Taken together,we investigated the mechanism of Brucella melitensis remodeling RAW264.7 cells activities through mi RNA at post-transcription level,and established base-editing method based on CRISPR/Cas9 system.This study provides an understanding and an effective genome editing platform for further study on Brucella gene function and pathogenic mechanism.The ongoing study would identify important genes in Brucella spp surviving during infection,which will provide insights into pathogenic mechanism and vaccine research.