Improvement to Gene Editing in Neospora Caninum: Knockout ku80 to Increase the Efficiency of Homologous Recombination

Abstract

Background: The CRISPR/Cas9 technology based on homologous recombination has been widely used for gene editing in organism. Gene deletion of ku80 complex can increase the efficiency of homologous recombination(HR), or gene editing by CRISPR/Cas9 in Arabidopsis thaliana and Toxoplasma gondii, which has remained elusive in Neospora caninum. Methods: Here, we knocked out the ku80 in Nc1 strain, named Δku80, using CRISPR/Cas9. In vitro, phenotype assays, including plaque formation, invasion, replication and egress were carried out to determination of the growth of Δku80. In vivo, we respectively injected BALB/c mice with different doses of NcKU80 and Nc1, and measured the survival curves and parasites burdens of mice infected. To test the efficiency of HR, Δku80 and Nc1 strains were transfected with the same HA-tagged plasmids, and the percentage of parasites with HA-tagged was investigated.Results: It is showed that the growth and virulence of Δku80 kept unaffected, while the efficiency of gene targeting via double-crossover at several genetic loci was increased 2 to 3 fold in Δku80. Conclusions: It is concluded that Δku80 can be used as an effective strain for rapid gene editing in N. caninum.