Deleting ku80 improves the efficiency of targeted gene editing in Neospora caninum.

CRISPR/Cas9 technology has been widely used for gene editing in organisms. Gene deletion of the ku80/ku70 complex can improve the efficiency of gene replacement in Arabidopsis thaliana, Cryptococcus neoformans, and Toxoplasma gondii, which remained elusive in Neospora caninum. Here, we knock out the ku80 gene in Nc1 strain by using CRISPR/Cas9, detect the growth rate and virulence of NcΔku80. Then we compare the efficiency of gene replacements between NcΔku80 and Nc1 strains by transfected with the same HA-tagged plasmids, and the percentage of HA-tagged parasites was investigated by IFA. The results showed that gene targeting efficiency was increased in the NcΔku80 strain via double crossover at several genetic loci, but its growth rate and virulence were unaffected. In conclusion, the NcΔku80 strain can be used as an effective strain for rapid gene editing of N. caninum.