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Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressing P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque\/human host cell tropism of P. knowlesi. Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.<\/jats:p>","DOI":"10.7554\/elife.45829","type":"journal-article","created":{"date-parts":[[2019,6,13]],"date-time":"2019-06-13T12:00:20Z","timestamp":1560427220000},"source":"Crossref","is-referenced-by-count":86,"title":["Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research"],"prefix":"10.7554","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4726-6693","authenticated-orcid":true,"given":"Franziska","family":"Mohring","sequence":"first","affiliation":[{"name":"Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melissa Natalie","family":"Hart","sequence":"additional","affiliation":[{"name":"Faculty of Infectious and 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