Development of a CRISPR/Cas9 system against ruminant animal brucellosis.

ABSTRACT

BACKGROUND: Brucellosis, caused by several Brucella species, such as the bacterium Brucella melitensis, is considered one of the most severe zoonotic diseases worldwide. Not only does it affect ruminant animal populations, leading to a substantial financial burden for stockbreeders, but also poses severe public health issues. For almost four decades in southern Europe and elsewhere, eradication of the disease has been based on ambiguously effective programs, rendering massive sanitation of livestock urgent and indispensable. Gene therapy, which has been proved effective in the clinic, could possibly constitute an alternative option towards a permanent cure for brucellosis, by aiding in the deletion or inactivation of genes associated with the replication of Brucella within the host cells. RESULTS: We infected ovine macrophages with B.melitensis, to simulate the host cell/microorganism interaction in vitro, and transduced the infected cells with CRISPR/Cas9 lentiviral vectors that target Brucella's RNA polymerase subunit A (RpolA) or virulence-associated gene virB10 at a multiplicity of infection of 60. We demonstrate a significant decrease in the bacterial load per cell when infected cells are transduced with the RpolA vector and that the number of internalized brucellae per cell remains unaffected when macrophages are transduced with a conventional lentiviral vector expressing the green fluorescence protein, thus underlining the bactericidal effect of our CRISPR/Cas9 system. CONCLUSIONS: Pending in vivo verification of our findings, overall, these results may prove critical not only for the treatment of human brucellosis, but for other infectious diseases in general.