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A heterologous marker-free strategy, CRISPR/Cas9pyrR, was developed by co-editing the target gene while introducing pyrimethamine resistance mutations into pfdhfr-ts using separate plasmids and PYR pressure. Proof-of-principle generation of GFP-tagged PfGEX1 and PfNUP116 shows stage-specific localization in gametocytes and distinct perinuclear positioning for PfNUP116. Sequential engineering further expands the toolkit via additional selection and tagging of PfAP2-G and PfNUP313.",{"@graph":69,"@context":123},[70,84,106],{"@type":71,"itemListElement":72},"BreadcrumbList",[73,77,79,82],{"item":74,"name":75,"@type":76,"position":8},"https://docshare.wps.com","Home","ListItem",{"item":78,"name":9,"@type":76,"position":14},"https://docshare.wps.com/document/",{"item":80,"name":40,"@type":76,"position":81},"https://docshare.wps.com/document/research-report/",3,{"item":83,"name":65,"@type":76,"position":19},"https://docshare.wps.com/document/a-heterologous-marker-free-selection-approach-for-crisprcas9-based-gene-editing-in-the-malaria-parasite-plasmodium-falciparum/230355/",{"url":83,"name":65,"@type":85,"image":86,"author":91,"headline":65,"publisher":94,"fileFormat":97,"inLanguage":63,"description":67,"dateModified":98,"datePublished":99,"encodingFormat":97,"isAccessibleForFree":100,"interactionStatistic":101},"DigitalDocument",{"url":87,"@type":88,"width":89,"height":90},"https://docshare.wps.com/thumbnails/a-heterologous-marker-free-selection-approach-for-crisprcas9-based-gene-editing-in-the-malaria-parasite-plasmodium-falciparum/230355.png","ImageObject",300,407,{"name":92,"@type":93},"\tCallum ","Person",{"url":74,"name":95,"@type":96},"DocShare","Organization","application/pdf","2026-10-17","2026-09-10",true,{"@type":102,"interactionType":103,"userInteractionCount":105},"InteractionCounter",{"@type":104},"ViewAction",13,{"@type":107,"mainEntity":108},"FAQPage",[109,115,119],{"name":110,"@type":111,"acceptedAnswer":112},"What limitation of existing CRISPR/Cas9 approaches motivates CRISPR/Cas9pyrR?","Question",{"text":113,"@type":114},"Existing methods depend on a limited set of heterologous drug resistance markers for selecting transgenic parasites, restricting repeated rounds of genetic modification.","Answer",{"name":116,"@type":111,"acceptedAnswer":117},"How does CRISPR/Cas9pyrR enable marker-free selection in P. falciparum?",{"text":118,"@type":114},"It simultaneously edits the gene of interest and introduces PYR resistance-conferring mutations in the pfdhfr-ts locus using separate plasmids, so only parasites acquiring both plasmids survive under PYR pressure.",{"name":120,"@type":111,"acceptedAnswer":121},"What localization patterns were observed for the GFP-tagged proteins PfGEX1 and PfNUP116?",{"text":122,"@type":114},"PfGEX1-GFP specifically marks the nuclear envelope in gametocytes but not asexual blood stages. PfNUP116-GFP is undetectable in asexual parasites and instead localizes to a distinct perinuclear region in early gametocytes.","https://schema.org",{"og:url":83,"og:type":125,"og:title":65,"og:site_name":95,"og:description":67},"article",{"robots":127,"canonical":83},"index,follow",{"doc_id":129,"site_id":62},230355,1789049425,{"code":4,"msg":5,"data":132},{"doc_id":129,"user_id":133,"nickname":92,"user_avatar":134,"doc_module":4,"category_id":39,"category_name":40,"doc_title":65,"doc_description":67,"doc_content":135,"file_id":136,"file_url":137,"file_type":138,"file_size":139,"view_count":105,"is_deleted":4,"is_public":8,"is_downloadable":8,"audit_status":8,"page_count":140,"language":141,"language_code":63,"site_id":62,"html_lang":63,"table_of_contents":142,"faqs":143,"seo_title":144,"seo_description":67,"update_tm":130,"read_time":145,"preview_page_count":4},137451211410,"https://ap-avatar.wpscdn.com/avatar/2000bb0a9246f588df?x-image-process=image/resize,m_fixed,w_180,h_180&k=1786362646172706240","| Parasitology | Research Article  \nA heterologous marker-free selection approach for CRISPR/Cas9-based gene editing in the malaria parasite Plasmodium falciparum  \nEilidh Carrington,1,2 Daniel Ballmer,1,2 Igor Niederwieser,1,2 Basil T. Thommen,1,2 Nicolas M. B. Brancucci,1,2 Till S. Voss1,2  \nAUTHOR AFFILIATIONS See a􀂀liation list on p. 15.  \nABSTRACT CRISPR/Cas9-based gene editing of the malaria parasite Plasmodium falciparum has emerged as a transformative tool for advancing functional studies on parasite biology and identifying new therapeutic targets. Currently applied CRISPR/Cas9 methodologies depend on a limited set of heterologous drug resistance markers for the selection of transgenic parasites, which restricts the potential for iterative genetic modi􀂁cations. Here, we developed a heterologous marker-free CRISPR/Cas9 gene editing strategy (CRISPR/Cas9pyrR) for P. falciparum based on the simultaneous editing of a gene of interest and introduction of pyrimethamine (PYR) resistance-conferring mutations into the dihydrofolate reductase-thymidylate synthase (pfdhfr-ts) gene. By providing a pfdhfrpyrR donor sequence and the Cas9 expression cassette on separate plasmids, CRISPR/Cas9pyrR ensures that only parasites acquiring both plasmids survive under PYR pressure. As a proof of principle, we applied CRISPR/Cas9pyrR to generate two transgenic parasite lines expressing GFP-tagged versions of the putative nuclear envelope protein PfGEX1 and nuclear pore protein PfNUP116, respectively. We show that PfGEX1-GFP marks the nuclear envelope speci􀂁cally in gametocytes, but not in asexual blood stage parasites. Similarly, and against previous reports, we 􀂁nd PfNUP116-GFP expression is undetectable in asexual parasites but instead localizes to a distinct perinuclear region in early gametocytes. These results suggest dynamic compositional changes of the nuclear periphery during sexual di􀂂erentiation. We further demonstrate sequential genetic engineering of the PfNUP116-GFP-expressing line using the human dhfr drug resistance marker combined with WR99210-based selection by additionally tagging PfAP2-G, the master transcriptional regulator of sexual commitment, and the nuclear pore protein PfNUP313 . Hence, CRISPR/Cas9pyrR provides a versatile and e􀂂ective new method that enhances and complements the current genetic toolkit for malaria research.  \nIMPORTANCE Malaria tropica, which is caused by the unicellular parasite Plasmodium falciparum, is one of the most devastating infectious diseases worldwide. The development of urgently needed e􀂂ective vaccines and new antimalarial drugs with novel modes of action requires a profound understanding of parasite biology. CRISPR/ Cas9-based genome engineering is beyond doubt the most important experimental approach to study the function and essentiality of parasite proteins and to identify and validate new vaccine and drug targets. In this study, we developed and successfully applied a modi􀂁ed CRISPR/Cas9 strategy, termed CRISPR/Cas9pyrR, that avoids the use of a heterologous drug resistance marker for the selection of genetically modi􀂁ed parasites. CRISPR/Cas9pyrR thus complements the CRISPR/Cas9 toolbox available for gene editing in P. falciparum and overcomes some of the limitations of currently employed protocols.  \nKEYWORDS malaria, Plasmodium falciparum, CRISPR/Cas9  \nEditor Giel G. van Dooren, Australian National University, Canberra, ACT, Australia  \nAddress correspondence to Till S. Voss, [till.voss@swisstph.ch](till.voss@swisstph.ch).  \nThe authors declare no con􀂀ict of interest.  \nSee the funding table on p. 15.  \nReceived 13 December 2025  \nAccepted 19 February 2026  \nPublished 26 March 2026  \nCopyright © 2026 Carrington et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.  \nMonth XXXX Volume 0 Issue 0 10. 1128/msphere.00884-25 1  \nDownloaded from [https://journals.asm.org/journal/msphere on 03 April 2026 by 2c0f:","cbCaifvTk99ZT6ad","https://ap.wps.com/l/cbCaifvTk99ZT6ad","pdf",448692,18,"English","# Abstract\n## Method: CRISPR/Cas9pyrR marker-free selection strategy\n## Proof of principle: GFP-tagged lines and localization\n## Sequential genetic engineering and expanded toolkit\n# Importance\n## Need for new targets and limitations of existing protocols","[{\"question\":\"What limitation of existing CRISPR/Cas9 approaches motivates CRISPR/Cas9pyrR?\",\"answer\":\"Existing methods depend on a limited set of heterologous drug resistance markers for selecting transgenic parasites, restricting repeated rounds of genetic modification.\"},{\"question\":\"How does CRISPR/Cas9pyrR enable marker-free selection in P. falciparum?\",\"answer\":\"It simultaneously edits the gene of interest and introduces PYR resistance-conferring mutations in the pfdhfr-ts locus using separate plasmids, so only parasites acquiring both plasmids survive under PYR pressure.\"},{\"question\":\"What localization patterns were observed for the GFP-tagged proteins PfGEX1 and PfNUP116?\",\"answer\":\"PfGEX1-GFP specifically marks the nuclear envelope in gametocytes but not asexual blood stages. PfNUP116-GFP is undetectable in asexual parasites and instead localizes to a distinct perinuclear region in early gametocytes.\"}]","A heterologous marker-free selection approach for CRISPR/Cas9-based gene editing in the malaria parasite Plasmodium falciparum | PDF",45]