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disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites\n                    <jats:italic>Trypanosoma congolense<\/jats:italic>\n                    and\n                    <jats:italic>T<\/jats:italic>\n                    .\n                    <jats:italic>vivax<\/jats:italic>\n                    , but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the\n                    <jats:italic>T<\/jats:italic>\n                    .\n                    <jats:italic>congolense<\/jats:italic>\n                    genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses\n                    <jats:italic>T<\/jats:italic>\n                    .\n                    <jats:italic>congolense-<\/jats:italic>\n                    optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides &gt;40,000 independent stable clones per transfection\u2013enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of\n                    <jats:italic>T<\/jats:italic>\n                    .\n                    <jats:italic>congolense<\/jats:italic>\n                    are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change\n                    <jats:italic>T<\/jats:italic>\n                    .\n                    <jats:italic>congolense<\/jats:italic>\n                    from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite.\n                  <\/jats:p>","DOI":"10.1371\/journal.ppat.1009224","type":"journal-article","created":{"date-parts":[[2021,1,22]],"date-time":"2021-01-22T17:56:29Z","timestamp":1611338189000},"page":"e1009224","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":31,"title":["Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo"],"prefix":"10.1371","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6572-0226","authenticated-orcid":true,"given":"Georgina","family":"Awuah-Mensah","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2208-1246","authenticated-orcid":true,"given":"Jennifer","family":"McDonald","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3677-5898","authenticated-orcid":true,"given":"Pieter C.","family":"Steketee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Delphine","family":"Autheman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Whipple","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9630-8733","authenticated-orcid":true,"given":"Simon","family":"D'Archivio","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cordelia","family":"Brandt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simon","family":"Clare","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katherine","family":"Harcourt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0537-0863","authenticated-orcid":true,"given":"Gavin 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