AUTHOR=Rico-San Román Laura , Hänggeli Kai Pascal Alexander , Hemphill Andrew , Horcajo Pilar , Collantes-Fernández Esther , Ortega-Mora Luis Miguel , Boubaker Ghalia 

TITLE=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

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=14

YEAR=2024

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2024.1419209

DOI=10.3389/fcimb.2024.1419209

ISSN=2235-2988

ABSTRACT=<p>As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in <italic>Neospora caninum</italic>, 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 (<italic>mdhfr-ts</italic>) 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 <italic>mdhfr-ts</italic> copies into other sites of the genome. To determine the number of integrated <italic>mdhfr-ts</italic> in <italic>N. caninum</italic>, 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 <italic>wtdhfrs-ts</italic> gene, as well as the mutated <italic>mdhfrs-ts</italic> present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic <italic>NC5</italic> gene. Thus, the <italic>dhfr-ts</italic> to <italic>NC5</italic> ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated <italic>mdhrf-ts</italic> gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the <italic>Neospora</italic> KO strains Nc<italic>ΔGRA7</italic> and Nc<italic>ΔROP40</italic>. For Nc<italic>ΔGRA7</italic>, the number of tachyzoites determined by <italic>dhfr-ts</italic> quantification was twice the number of tachyzoites determined by <italic>NC5</italic> quantification, thus indicating that only one <italic>mdhfr-ts</italic> copy was integrated. The results obtained with the Nc<italic>ΔROP40</italic> strain, however, showed that the number of <italic>dhfr-ts</italic> copies per genome was substantially higher, indicating that at least three copies of the selectable <italic>mdhfr-ts</italic> 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 <italic>N. caninum</italic> strains.</p>