Immunization with the NcMYR1 gene knockout strain effectively protected C57BL/6 mice and their pups against the challenge.

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 virulence will provide potential candidates for vaccine development against neosporosis. In the present study, 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 CRISPR-Cas9 gene knockout strain as an attenuated vaccine. NcMYR1 was determined to be a cytoplasmic protein in 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 challenge. Above all, NcMYR1 is a virulence factor and the ΔNcMYR1-1 strain could be used as a candidate vaccine against infection and vertical transmission.