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Due to the limited effectiveness of currently available drugs, there is an urgent need for new therapeutic approaches to control neosporosis. Luciferase-based assays are potentially powerful tools in the search for antiprotozoal compounds, permitting the development of faster and more automated assays. The aim of this study was to construct a luciferase-expressing\n                      <jats:italic>N. caninum<\/jats:italic>\n                      and evaluate anti-\n                      <jats:italic>N. caninum<\/jats:italic>\n                      drugs.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Luciferase-expressing\n                      <jats:italic>N. caninum<\/jats:italic>\n                      (Nc1-Luc) was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR\/Cas9). After testing the luciferase expression and phenotype of the Nc1-Luc strains, the drug sensitivity of Nc1-Luc strains was determined by treating them with known positive or negative drugs and calculating the half-maximal inhibitory concentration (IC\n                      <jats:sub>50<\/jats:sub>\n                      ). The selective pan-rapidly accelerated fibrosarcoma (pan-RAF) inhibitor TAK-632 was then evaluated for anti-\n                      <jats:italic>N. caninum<\/jats:italic>\n                      effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo studies.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parental strains Nc1, and Nc1-Luc strains can be used to determine the IC\n                      <jats:sub>50<\/jats:sub>\n                      for anti-\n                      <jats:italic>N. caninum<\/jats:italic>\n                      drugs. Using the Nc1-Luc strains, TAK-632 showed promising activity against\n                      <jats:italic>N. caninum<\/jats:italic>\n                      , with an IC\n                      <jats:sub>50<\/jats:sub>\n                      of 0.6131\u00a0\u03bcM and a selectivity index (SI) of 62.53. In vitro studies demonstrated that TAK-632 inhibited the invasion, proliferation, and division of\n                      <jats:italic>N. caninum<\/jats:italic>\n                      tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of\n                      <jats:italic>N. caninum<\/jats:italic>\n                      in mice and significantly reduced the parasite burden in the brain.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      In conclusion, a luciferase-expressing\n                      <jats:italic>N. caninum<\/jats:italic>\n                      strain was successfully constructed, which provides an effective tool for drug screening and related research on\n                      <jats:italic>N. caninum<\/jats:italic>\n                      . In addition, TAK-632 was found to inhibit the growth of\n                      <jats:italic>N. caninum<\/jats:italic>\n                      , which could be considered as a candidate lead compound for new therapeutics for neosporosis.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Graphical Abstract<\/jats:title>\n                  <\/jats:sec>","DOI":"10.1186\/s13071-024-06195-8","type":"journal-article","created":{"date-parts":[[2024,3,8]],"date-time":"2024-03-08T06:01:46Z","timestamp":1709877706000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Construction of luciferase-expressing Neospora caninum and drug screening"],"prefix":"10.1186","volume":"17","author":[{"given":"Fei","family":"Wang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yangfei","family":"Xue","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanqun","family":"Pei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meng","family":"Yin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhepeng","family":"Sun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zihui","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qun","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,3,8]]},"reference":[{"key":"6195_CR1","doi-asserted-by":"publisher","first-page":"323","DOI":"10.1128\/CMR.00031-06","volume":"20","author":"JP Dubey","year":"2007","unstructured":"Dubey JP, Schares G, Ortega-Mora LM. 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