Efficient Gene Replacements in <i>Toxoplasma gondii</i> Strains Deficient for Nonhomologous End Joining

<jats:title>ABSTRACT</jats:title> <jats:p> A high frequency of nonhomologous recombination has hampered gene targeting approaches in the model apicomplexan parasite <jats:italic>Toxoplasma gondii</jats:italic> . To address whether the nonhomologous end-joining (NHEJ) DNA repair pathway could be disrupted in this obligate intracellular parasite, putative KU proteins were identified and a predicted <jats:italic>KU80</jats:italic> gene was deleted. The efficiency of gene targeting via double-crossover homologous recombination at several genetic loci was found to be greater than 97% of the total transformants in <jats:italic>KU80</jats:italic> knockouts. Gene replacement efficiency was markedly increased (300- to 400-fold) in <jats:italic>KU80</jats:italic> knockouts compared to wild-type strains. Target DNA flanks of only ∼500 bp were found to be sufficient for efficient gene replacements in <jats:italic>KU80</jats:italic> knockouts. <jats:italic>KU80</jats:italic> knockouts stably retained a normal growth rate in vitro and the high virulence phenotype of type I strains but exhibited an increased sensitivity to double-strand DNA breaks induced by treatment with phleomycin or γ-irradiation. Collectively, these results revealed that a significant KU-dependent NHEJ DNA repair pathway is present in <jats:italic>Toxoplasma gondii</jats:italic> . Integration essentially occurs only at the homologous targeted sites in the <jats:italic>KU80</jats:italic> knockout background, making this genetic background an efficient host for gene targeting to speed postgenome functional analysis and genetic dissection of parasite biology. </jats:p>