TaqMan-quantitative PCR assays applied in Neospora caninum knock-outs generated through CRISPR-Cas9 allow to determine the copy numbers of integrated dihydrofolate reductase-thymidylate synthase drug selectable markers

As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in , an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase ( ) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple copies into other sites of the genome. To determine the number of integrated in , a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy gene, as well as the mutated present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic gene. Thus, the to ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the KO strains Nc and Nc . For Nc , the number of tachyzoites determined by quantification was twice the number of tachyzoites determined by quantification, thus indicating that only one copy was integrated. The results obtained with the Nc strain, however, showed that the number of copies per genome was substantially higher, indicating that at least three copies of the selectable marker were integrated into the genomic DNA during gene editing by CRISPR-Cas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT strains.