Immunization with the NcMYR1 gene knockout strain effectively protected C57BL/6 mice and their pups against the <i>Neospora caninum</i> challenge

<i>Neospora caninum</i> is an important protozoan parasite that causes abortion in cattle and nervous system dysfunction in dogs. No effective drugs and vaccines for neosporosis are available. Further elucidation of proteins related to <i>N. caninum</i> virulence will provide potential candidates for vaccine development against neosporosis. In the present study, <i>N. caninum</i> c-Myc regulatory protein (NcMYR1) gene knockout strains (ΔNcMYR1-1, ΔNcMYR1-2, and ΔNcMYR1-3) were generated using the CRISPR-Cas9 gene editing system to investigate phenotype changes and the potential of the ΔNcMYR1-1 strain as an attenuated vaccine, and this is the first time of using the <i>N. caninum</i> CRISPR-Cas9 gene knockout strain as an attenuated vaccine. NcMYR1 was determined to be a cytoplasmic protein in <i>N. caninum</i> tachyzoites. The deficiency of NcMYR1 decreased the plaque area and the rate of invasion, replication, and egression of the parasites. ΔNcMYR1-1 strain-infected C57BL/6 mice had 100% survival rate, reduced parasite burden, and alleviated pathological changes in tissues compared with those in Nc-1 strain-infected mice. Immunization with ΔNcMYR1-1 tachyzoites increased the productions of cytokines in mice, with a survival rate reaching 80%, and the parasite burdens in the liver and spleen were greatly reduced when challenged with the Nc-1 strain with a lethal dose after 40 days of ΔNcMYR1-1 tachyzoite immunization. ΔNcMYR1 immunization could decrease the abortion rate of female mice from 71.4% to 12.5% and increase the survival rate of pups from 12.5% to 83.3% against the <i>N. caninum</i> challenge. Above all, NcMYR1 is a virulence factor and the ΔNcMYR1-1 strain could be used as a candidate vaccine against <i>N. caninum</i> infection and vertical transmission.