{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,15]],"date-time":"2026-04-15T13:23:56Z","timestamp":1776259436833,"version":"3.50.1"},"reference-count":49,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,4,15]],"date-time":"2026-04-15T00:00:00Z","timestamp":1776211200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Intramural Research Program of the National Institutes of Health"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Background: Malaria parasites import essential nutrients from plasma into their host erythrocytes through the plasmodial surface anion channel (PSAC), a conserved ion and nutrient channel on the infected cell surface. A parasite-encoded ternary complex consisting of CLAG3, RhopH2, and RhopH3 determines PSAC activity, but the precise contributions of each member to formation of the nutrient uptake pore remains uncertain. Methods: Here, we devised a two-step CRIPSR transfection strategy to examine an amphipathic CLAG3 helix, termed \u03b1-helix 44 (\u03b1-H44), as a candidate pore-lining domain. Results: A CLAG3 truncation protein without \u03b1-H44 phenocopies a CLAG3 knockout line, suggesting a critical role of \u03b1-H44 in formation of the nutrient channel; CLAG3 restoration using a recodonized \u03b1-H44 restores PSAC activity fully. A saturation mutagenesis library that splits the helix into four sequential segments was devised and implemented. Two engineered mutants exhibit distinct PSAC phenotypes; their cultures failed to expand in a modified medium that approximates in vivo nutrient availability. Conclusions: These studies support a \u03b1-H44 role in channel permeation and block by a strain-specific inhibitor. Our strategy will enable saturation mutagenesis to determine how PSAC achieves its unique ion and nutrient selectivity and should help guide drug discovery against this antimalarial target.<\/jats:p>","DOI":"10.3390\/genes17040462","type":"journal-article","created":{"date-parts":[[2026,4,15]],"date-time":"2026-04-15T12:25:30Z","timestamp":1776255930000},"page":"462","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A CRISPR-Based Mutagenesis Strategy for Examining CLAG3 Helix 44 Contribution to Malaria Parasite Nutrient Uptake Channels"],"prefix":"10.3390","volume":"17","author":[{"given":"Zabdi","family":"Gonzalez-Chavez","sequence":"first","affiliation":[{"name":"Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mansoor A.","family":"Siddiqui","sequence":"additional","affiliation":[{"name":"Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sundar","family":"Ganesan","sequence":"additional","affiliation":[{"name":"Biological Imaging Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2150-2483","authenticated-orcid":false,"given":"Sanjay A.","family":"Desai","sequence":"additional","affiliation":[{"name":"Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/bs.apar.2015.02.004","article-title":"PfEMP1\u2014A Parasite Protein Family of Key Importance in Plasmodium falciparum Malaria Immunity and Pathogenesis","volume":"88","author":"Hviid","year":"2015","journal-title":"Adv. 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