{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,23]],"date-time":"2026-08-23T10:38:48Z","timestamp":1787481528662,"version":"build-2736575974"},"reference-count":29,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,6,21]],"date-time":"2024-06-21T00:00:00Z","timestamp":1718928000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats: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 <jats:italic>Neospora caninum<\/jats: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 (<jats:italic>mdhfr-ts<\/jats: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 <jats:italic>mdhfr-ts<\/jats:italic> copies into other sites of the genome. To determine the number of integrated <jats:italic>mdhfr-ts<\/jats:italic> in <jats:italic>N. caninum<\/jats: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 <jats:italic>wtdhfrs-ts<\/jats:italic> gene, as well as the mutated <jats:italic>mdhfrs-ts<\/jats:italic> present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic <jats:italic>NC5<\/jats:italic> gene. Thus, the <jats:italic>dhfr-ts<\/jats:italic> to <jats:italic>NC5<\/jats:italic> ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated <jats:italic>mdhrf-ts<\/jats:italic> gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the <jats:italic>Neospora<\/jats:italic> KO strains Nc<jats:italic>\u0394GRA7<\/jats:italic> and Nc<jats:italic>\u0394ROP40<\/jats:italic>. For Nc<jats:italic>\u0394GRA7<\/jats:italic>, the number of tachyzoites determined by <jats:italic>dhfr-ts<\/jats:italic> quantification was twice the number of tachyzoites determined by <jats:italic>NC5<\/jats:italic> quantification, thus indicating that only one <jats:italic>mdhfr-ts<\/jats:italic> copy was integrated. The results obtained with the Nc<jats:italic>\u0394ROP40<\/jats:italic> strain, however, showed that the number of <jats:italic>dhfr-ts<\/jats:italic> copies per genome was substantially higher, indicating that at least three copies of the selectable <jats:italic>mdhfr-ts<\/jats: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 <jats:italic>N. caninum<\/jats:italic> strains.<\/jats:p>","DOI":"10.3389\/fcimb.2024.1419209","type":"journal-article","created":{"date-parts":[[2024,6,21]],"date-time":"2024-06-21T04:26:14Z","timestamp":1718943974000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":5,"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"],"prefix":"10.3389","volume":"14","author":[{"given":"Laura","family":"Rico-San Rom\u00e1n","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kai Pascal Alexander","family":"H\u00e4nggeli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew","family":"Hemphill","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pilar","family":"Horcajo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Esther","family":"Collantes-Fern\u00e1ndez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luis Miguel","family":"Ortega-Mora","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ghalia","family":"Boubaker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2024,6,21]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"102576","DOI":"10.1016\/j.parint.2022.102576","article-title":"Role of dense granule antigen 7 in vertical transmission of Neospora caninum in C57BL\/6 mice infected during early pregnancy","volume":"89","author":"Abdou","year":"2022","journal-title":"Parasitol. Int."},{"key":"B2","doi-asserted-by":"publisher","first-page":"177","DOI":"10.1186\/s13059-018-1534-x","article-title":"CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters","volume":"19","author":"Alkan","year":"2018","journal-title":"Genome Biol."},{"key":"B3","doi-asserted-by":"publisher","first-page":"597","DOI":"10.1016\/j.ijpara.2018.03.002","article-title":"Toxoplasma CRISPR\/Cas9 constructs are functional for gene disruption in","volume":"48","author":"Arranz-Sol\u00eds","year":"2018","journal-title":"Neospora caninum. Int. J. Parasitol."},{"key":"B4","doi-asserted-by":"publisher","first-page":"147","DOI":"10.1016\/j.ijpara.2013.08.002","article-title":"Identification of three novel Toxoplasma gondii rhoptry proteins","volume":"44","author":"Camejo","year":"2014","journal-title":"Int. J. Parasitol."},{"key":"B5","doi-asserted-by":"publisher","first-page":"1194","DOI":"10.1128\/JCM.40.4.1194-1198.2002","article-title":"Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time PCR","volume":"40","author":"Collantes-Fern\u00e1ndez","year":"2002","journal-title":"J. Clin. Microbiol."},{"key":"B6","doi-asserted-by":"publisher","first-page":"1119","DOI":"10.1017\/S003118201800001X","article-title":"New and emerging uses of CRISPR\/Cas9 to genetically manipulate apicomplexan parasites","volume":"145","author":"Di Cristina","year":"2018","journal-title":"Parasitology"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.3389\/fvets.2021.634612","article-title":"Disruption of dense granular protein 2 (GRA2) decreases the virulence of Neospora caninum","volume":"8","author":"Dong","year":"2021","journal-title":"Front. Vet. Sci."},{"key":"B8","doi-asserted-by":"crossref","DOI":"10.1201\/9781315152561","volume-title":"Neosporosis in Animals","author":"Dubey","year":"2017"},{"key":"B9","doi-asserted-by":"publisher","first-page":"90","DOI":"10.1016\/j.vetpar.2011.05.031","article-title":"Neosporosis in animals-The last five years","volume":"180","author":"Dubey","year":"2011","journal-title":"Vet. Parasitol."},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0271011","article-title":"Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9","volume":"17","author":"H\u00e4nggeli","year":"2022","journal-title":"PloS One"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.3389\/fvets.2023.1249410","article-title":"Bovine infectious abortion: a systematic review and meta-analysis","volume":"10","author":"Hecker","year":"2023","journal-title":"Front. Vet. Sci."},{"key":"B12","doi-asserted-by":"publisher","first-page":"1562","DOI":"10.1021\/acs.jmedchem.8b01754","article-title":"Discovery of selective Toxoplasma gondii dihydrofolate reductase inhibitors for the treatment of toxoplasmosis","volume":"62","author":"Hopper","year":"2019","journal-title":"J. Med. Chem."},{"key":"B13","doi-asserted-by":"publisher","first-page":"816","DOI":"10.1126\/science.1225829","article-title":"A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity","volume":"337","author":"Jinek","year":"2012","journal-title":"Science"},{"key":"B14","doi-asserted-by":"publisher","first-page":"7473","DOI":"10.1093\/nar\/gku402","article-title":"CRISPR\/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences","volume":"42","author":"Lin","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"B15","doi-asserted-by":"publisher","first-page":"205","DOI":"10.1016\/j.cvfa.2019.11.004","article-title":"Neosporosis, toxoplasmosis, and sarcocystosis in ruminants: an update","volume":"36","author":"Lindsay","year":"2020","journal-title":"Veterinary Clinics North America - Food Anim. Pract."},{"key":"B16","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1016\/0378-1119(90)90145-H","article-title":"Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions","volume":"93","author":"Longo","year":"1990","journal-title":"Gene"},{"key":"B17","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1128\/AEM.01350-18","article-title":"Neospora caninum dense granule protein 7 regulates the pathogenesis of neosporosis by modulating host immune response","volume":"84","author":"Nishikawa","year":"2018","journal-title":"Appl. Environ. Microbiol."},{"key":"B18","doi-asserted-by":"publisher","first-page":"4067","DOI":"10.1016\/j.cell.2022.09.026","article-title":"Structural basis for Cas9 off-target activity","volume":"185","author":"Pacesa","year":"2022","journal-title":"Cell"},{"key":"B19","doi-asserted-by":"publisher","first-page":"41","DOI":"10.1186\/1297-9716-42-41","article-title":"In vitro invasion efficiency and intracellular proliferation rate comprise virulence-related phenotypic traits of Neospora caninum","volume":"42","author":"Regidor-Cerrillo","year":"2011","journal-title":"Vet. Res."},{"key":"B20","doi-asserted-by":"publisher","first-page":"133","DOI":"10.1016\/j.ijpara.2012.10.022","article-title":"What is the global economic impact of Neospora caninum in cattle - The billion dollar question","volume":"43","author":"Reichel","year":"2013","journal-title":"Int. J. Parasitol."},{"key":"B21","doi-asserted-by":"publisher","first-page":"e1002567","DOI":"10.1371\/journal.ppat.1002567","article-title":"Comparative genomics of the apicomplexan parasites Toxoplasma gondii and Neospora caninum: Coccidia differing in host range and transmission strategy","volume":"8","author":"Reid","year":"2012","journal-title":"PloS Pathog."},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1016\/j.vetpar.2023.109973","article-title":"Characterization of Neospora caninum virulence factors NcGRA7 and NcROP40 in bovine target cells","volume":"320","author":"Rico-San Rom\u00e1n","year":"2023","journal-title":"Vet. Parasitol."},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.3390\/pathogens11090998","article-title":"NcGRA7 and ncROP40 play a role in the virulence of Neospora caninum in a pregnant mouse model","volume":"11","author":"Rico-San Rom\u00e1n","year":"2022","journal-title":"Pathogens"},{"key":"B24","doi-asserted-by":"publisher","first-page":"59","DOI":"10.1016\/S0968-0896(02)00325-5","article-title":"Synthesis of new 2,4-Diaminopyrido[2,3-d]pyrimidine and 2,4-Diaminopyrrolo[2,3-d]pyrimidine inhibitors of Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium dihydrofolate reductase","volume":"11","author":"Rosowsky","year":"2003","journal-title":"Bioorg Med. Chem."},{"key":"B25","doi-asserted-by":"publisher","first-page":"701","DOI":"10.1016\/j.ijpara.2017.08.002","article-title":"Advances in the application of genetic manipulation methods to apicomplexan parasites","volume":"47","author":"Suarez","year":"2017","journal-title":"Int. J. Parasitol."},{"key":"B26","doi-asserted-by":"publisher","first-page":"197","DOI":"10.1007\/s00436-020-06961-4","article-title":"Function of Neospora caninum dense granule protein 7 in innate immunity in mice","volume":"120","author":"Wang","year":"2021","journal-title":"Parasitol. Res."},{"key":"B27","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s13071-021-05023-7","article-title":"Biotinylation of the Neospora caninum parasitophorous vacuole reveals novel dense granule proteins","volume":"14","author":"Yang","year":"2021","journal-title":"Parasit Vectors"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1038\/mtna.2015.37","article-title":"Off-target effects in CRISPR\/cas9-mediated genome engineering","volume":"4","author":"Zhang","year":"2015","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.3389\/fvets.2020.562730","article-title":"Effects of dense granular protein 6 (GRA6) disruption on Neospora caninum virulence","volume":"7","author":"Zhao","year":"2020","journal-title":"Front. Vet. Sci."}],"container-title":["Frontiers in Cellular and Infection Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2024.1419209\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,6,21]],"date-time":"2024-06-21T04:26:17Z","timestamp":1718943977000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2024.1419209\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,6,21]]},"references-count":29,"alternative-id":["10.3389\/fcimb.2024.1419209"],"URL":"https:\/\/doi.org\/10.3389\/fcimb.2024.1419209","relation":{},"ISSN":["2235-2988"],"issn-type":[{"value":"2235-2988","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,6,21]]},"article-number":"1419209"}}